Transplant Immunology 1998; 6: 193-197

Genotyping for polymorphisms in

interferon-y, interleukin-10, transforming
growth factor+1 and tumour necrosis
factora genes: a technical report
Chris Perrey, Vera Pravica, Paul J Sinnott and Ian V Hutchinson
School of Biological Sciences, University of Manchester; Manchester
Received 6 April 1998; accepted 21 April 1998

Abstract: Polymorphic variants of cytokine genes are associated with acute and chronic transplant rejec-
tion. In this technical report, the methods currently used in our centre to genotype individuals for inter-
feron-y, interleukin-10, transforming growth factor-b1 and tumour necrosis factor-u are described in detail.
The DNA sequences of primers and probes, and conditions for polymerase chain reactions are given, and
the allele and genotype frequencies in our control populations are summarized.

Introduction development and will be reported elsewhere, as will the method-

Allelic variants of cytokine genes are associated with higher or
lower production of cytokines, both in vitrr? and in vivo.' We
have shown that the genotype encoding high production of
tumour necrosis factor-a (TNF-a) is articularly associated with
acute rejection of heart?’ kidne $ ,’ and liver” transplants.
Paradoxically, the high producer allele of interleukin-10 (IL-lo)
is associated with protection against rejection of transplanted
hearts”’ but is associated with worse acute rejection of kidney
transplants.g Interferon? (EN-y) genotype is related to acute
kidney graft rejection” and to the development of fibrosis in
lung all~grafts.‘~ FinaUy, transkrrnkg grcnvth f&tor_B (TGF-
pl) genotype is signikmtly associated with fibr&j and chron-
ic rejection of lung tranuplants3~‘2 and may have a bearing on
chronic rejection of kidney &too.
The important porymorphirianslie in the promoter reqions of
IL-lo’ and TNF-a,” in the first intron of the IFN-y gene and in
the signal sequence of TGF-p1.3 This technical paper summarizes
the methods we currently use to type polymorphisms in these four
cytokine genes. These involve the polymerase chain reaction
(PCR) and either sequence-spec& o&x&e&& probing or
the determination of alleles -ding to t&e size of the PCR
product by gel electrophoresis. A&native methods are under
Address for correspondence: IV Hut&bon, Immunology Research
Group, School of Biological Sciences, University of Manchester,
Stopford Building 3.239, Oxford Road, Manchester Ml3 9PT, UK E-
mail: Ian.Hutchinson@man.ac.uk
0 Arnold 1998
ology for the typing of other cytokine gene polymorphisms.
Methods
DNA extraction
For this study, genomic DNA from whole blood or frozen cells
was obtained by phenol extraction and ethanol precipitation
following proteinase K (Boehringer Mannheim, Lewes, UK)
digestion. Commercially available DNA extraction kits have
been used suaxssWy in other studies.
DNA was ampWed using PCR performed on a PTC-100 ther-
mal cycler (Genetic &search Instrumentation Ltd, Dunmoor,
UK). Each 30 @ rcxtion n&ure contained 2 pl of test DNA
(50-200 ng), 50 mM KCl, 10 mM ‘I&HCl, 0.1% lfiton X-100,
200 pM each dATp dm, dGTP and dTTP (Gibco BFU), 2.5
mM MgClz (except for TGF-pl where 1.5 mM MgCl, was used),
1 M betaine mono&We @@a, Poole, Dorset, UK), 0.5 pM
each primer and 1 U ?&g p@mcmse (Gibco BRL, Paisley, UK).
After an initial time of 5 min, samples were subjected
to 30 rounds at !XPCfar 30 8, @mealing temperature (depend-
ingonthesetofprimcns)fbr3Osand724Iforlmin,witha
fmal extension time of5 min at 72°C. The primers used in each
reaction and their anneaiing temperatures are shown in Table
1. The amplified products were monitored by electrophoresis on
a 2% agarose gel with ethidium bromide (10 mg/l).
0966_3274(98)TI225OA
194 C A3rey et al.

‘Ihble 1 Primers and annealing temperatures for cytokine gene polymorphisms

Primer pairs Sequence T (“C)

TGF-Sl sense S-CITCACCAGCTCCATGTCGATAG-3 60
TGF-j31 antisense 5’-ACTGCGCCCPK’ICCCTG-3
TNF-a -308 sense 5’-ACTCAACACAGCITIT CCCTCCA-3 66
TNF-a -308 antisense 5’-TCCTCCCI’GCTCCGATTCCG-3
IL-10 sense 5’-ATCCAAGACAACACTACTAA-3 55.5
IL-10 antisense 5’-TAAATATCCC-3
IPN-y sense 5’-GCTGTCAT~TAATA’ITCAGAC-3 56
IFN-yantisense 15’~CGAGClT&MAGATAGITCG3’
Regions amplified: TGF-01: +691 to +%5, W-a; -331 to -224, IL-l& -1115 to -528, IFN-T +812 to +932.

TN&, IL10 and TGF-@l promoter region
polymorphism analysis
Sequence-speci$c oligonucleotide pminhg (SOP). After specific
PCR reactions were performed, two 5’ biotinylated oligo-
nucleotide probes (Genosys, UK, Pampisford, UK) were used to
positively identify each polymorphism in cytokine gene promot-
ers by a dot blot technique. A total of 2 pl of PCR product was
blotted onto Hybond N+ nylon transfer membrane (Amersham
International, Slough, UK). The double-stranded DNA was sep
arated by treating membranes with denaturing solution (0.5 M
NaOH and 1.5 M NaCI) for 5 mm and then neutralizing solution
(1.5 M NaCl and 0.5 M Tiis, pH 7.5) for 1 min. The membranes
were baked in an oven at WC for 10 min and the DNA was immo-
biied onto membrane by crosshnking in a UV Stratalinker
(Stratagen Ltd, Cambridge, UK) at 120 mJ/cm2. The membranes
were incubated in 50 ml tubes (Falcon, Becton Dickinson, New
Jersey, USA) with 10 ml of 5x SSC hybridization buffer (where
5x SSC is 0.75 M NaCl and 0.075 M sodium citrate) with 0.5%
blocking agent (milh powder), 0.1% ZV-lamylsarcosine and 0.02%
sodium dodecyl sulphate (SDS) for 30 min at 42.5”C. Following
the addition of 400 ng of a specific probe (see Table 2) to each tube,
membranes were ahowed to hybridize for 90 min at 42.5”C. The
membranes were washed twice in 5x SSC containing 0.1% SDS
for 5 min at room temperature and then stringency washed in lx

lbble 2 Probes and stringency temperatures used in cytokine gene polymorphisms

Probe Seauence T (“0

TGF-Bl codon lO*C coding strand 5’-GCTGCTGC~GCTGCTGC-3 58
TGF-pl codon lO*T coding strand 5’-GCTGCTG~GCTGCTGCT-3’
TGF-Sl codon 25*G coding strand 5’-GCCTGGCC($GCCGGCCG-3’ 62
TGF-or codon 25*C coding strand S-GCCTGGCC~GCCGGCCG-3
TN&a: -308*G coding strand 5’-GAGGGGCATGQGGACGG-3 56
TN&a: -308*A noncoding strand 5’-CCCGTC~CATGCCCCTC-3 59
IL-10 -1082”G coding strand 5’~lTCl-TTGGGA~GGGGAAG-3 47
IL-10 -1082*A noncoding strand 5’-ACITCCC~CCCAAAGAA-3
IL-10 -819*C coding strand 5’-CAGGTGATGTAACATCETGTGC-3 61
IL-10 -819*T noncoding strand 5’-GCACAGAGATA~ACATCACCKZ3
IL-10 -592*C coding strand S-CCGCCTGT~CT~AGGAA-3 48.5
IL-10 -592*A noncoding strand S-TTCCTACAG~ACAGGCGGG-3

Codon 10 Coaon ZU

F‘lgure 1 Autoradiography of TGF-& hybridization probes. Poiymerase chain reaction products from individuals were placed in the same position
on each membrane. Photographs show positive results for C and/or T for codon 10 and G and/or C for codon 25, respectively.

ltansplant immunology 1998; 6: 193-197


2f3 315 3/3 2f2 3/3 a3 m
Figure 2 Separation of the polymerase chain reaction products containing polymorphic dinucleotide repeat on the polyacrylamide gel. Allele #2
has 12 CA repeats, #3 has 13, #4 has 14 and #5 has 15 CA repeats.
SSC with 0.1% SDS for 30 min at the temperature appropriate for
each cytokine, as stated in ‘Iable 2. The membranes were washed
for 1 min in 0.15 M NaCl and 0.1 M ‘Iris buffer, pH 7.5, incubat-
ed for 30 min at room temperature in the same buffer containing
0.5% milk powder, as a blocking agent to reduce nonspecific bind-
ing, and then incubated with strept peroxidase
conjugate (Amersham International, Slough, UK) for 30 min at
room temperature before detection by chemiluminescence using
the ECLR system (Amersham International, Slough, UK). X-ray
llhns were exposed and binding of allele-specific probes was used
to determine genotypes (Figure 1).
Analysjs of microsatelIite polymorphism in the first
intron of IF3I-y gene
After specific PCR reactions were performed, 8 pl of each sam-
ple was mixed with 3 pl of 5x ‘Iris-borate electrophoresis load-
ing buffer (Tris 0.89 M, Boric acid 0.89 M, EDTA 0.02 M, pH
8, 49% glycerol, 0.1% SDS, 1% bromophenol blue) and sepa-
rated by polyacrylamide gel electrophoresis (PAGE) (using a gel
containing 12% acrylamidebis acryhunide, 19:l) at 35 mA for
4 h. Following electrophoresis, gels were stained with ethidium
bromide (10 mg/l), visualized with UV light on a transillumina-
tor and photographed for the definition of alleles (Figure 2).
Results and discussion
The methods describe here have been used successfully to type
cytokine gene polymorphisms for interferon-y, IL-IO, TNF-cz
and TGF-81, cytokines known to be associated with acute and
chronic human organ graft rejection.5-‘3
An example of the PCR-SSOP results of the detection of
polymorphisms in the TGF-81 leader sequence is shown in
Figure 1. The dot in each corresponding position on all four
membranes represents the DNA from an individual. Hence, the
individual whose DNA is at the top right-hand comer is codon
10 *C positive and *T negative (i.e. homozygous C/C) and
codon 25 *G positive and *C positive (i.e. heteroxygous G/C).
The PCR-SSOP method has the advantages that large batches
(40-80) can be done at one time, and that alleles are positively
identified by the binding of specific probes so that homoxygotes
can be defined with certainty. However, the PCR-SSOP
Transplant Immunology 1998; 6: 193-197
Genotypingfor polymorphisms: a technical report 195
30 2f3 3f4 3/4 313 2/s z4 2i3
approach is cumbersome for individual samples, and for this
reason we are developing amplification refractory mutation sys-
tem (ARMS)-PCR methodologies for genotyping.
The identification of the IFN-y CA-repeat polymorphisms are
shown in Figure 2. A single band represents a putative homoxy-
gote, so that the sample fourth from the left is a #2/#2 homozy-
gote and the third sample, with a slower band, is a #3/#3
homoxygote. Heterozygotes have two bands, as illustrated in the
three samples on the right, which are identified as having alle-
les #2l#3, #2/#4 and #2/#5, respectively (see Figure 2). An
example of the band identifying allele #l, which is very rare and
runs below the allele #2 band, is not shown on this gel. At pre-
sent, we have no simple manual alternative to the PAGE
approach to determine the number of CA repeats in the first
intron of the IFN-7gene. This method allowed us to type up to
30 samples.
The distribution of alleles and genotypes in our control pop-
ulations are shown in Tables 3-6. It should be noted that these
are principally Caucasian healthy volunteers or cadaveric renal
transplant donors. Any study should have a set of matched con-
trols, particularly in studies of other ethnic groups. We have rea-
son to suggest that allele distribution may be quite different in
other populations.
In general terms, we can ascribe high, intermediate and low
cytokine-producer status according to zygosity, being homozy-
gotes high/high, heteroxygotes high/low and homozygotes
low/low, respectively. The high responder alleles are TNF-a -
308 *A; TGF-81 codon 10 *T (Leu), codon 25 *G (Arg); IFN-
y allele #2; IL-10 -1082 *G. These are indicated in Tables 3-6,
and have a bearing on the interpretation of cytokine gene inher-
itance in transplant patients.
It is worth emphasizing that the cytokine gene polymorphisms
described in this technical paper are known to have functional
relevance, either because they directly affect gene transcription
or protein synthesis, or because they are in very close linkage
with a functional mutation. In particular, they are strongly sta-
tistically associated with post-transplant outcome, and may serve
as useful prognostic indicators of transplant rejection and
immunosuppression.
196 C l+rrey et al.

lhble 3 Frequences of TGF-gl genotypes for codon 10 and codon 25 and haplotype inheritance in corn&r

TGF-/31 poiymorphisms Genotype ControIs (n = 107) Frequency (%)

Ahele (phenotype)
Codon lO*Leu (high) T 139 65
Codon lO*Pro (low) ,C 75 35
Codon 25*Arg (high) G 193 90
Codon 25*Pro (low) C 21 10
Genotype (phenotype)
Codon 10
Leu/Leu (high) Tn. 44 41
Leu/Pro (intermediate) CfI 51 48
Pro/Pro (low) c/c 12 11
Codon 25
Arg/Arg (high) G/G a7 81
ArgRro (intermediate) G/C 19 18
Pro/Pro (low) c/c 1 1
Haplotype inheritance
LeuiLeu ArgMrg “r/T G/G 44 41
LeuiPro ArglArg “TIC G/G 38 36
Leu/Pro Arg/Pro bT/C G/C 13 12
Pro/Pro Argkg bC/C G/G 5 5
LeulLeu ArgPro t’IR G/C 0 0
Pro/Pro Arg/Pro “C/C G/C 6 6
Pro/Pro Pro/Pro ‘C/C G/C 1 1
LeulLeu Pro/Pro WT c/c 0 0
Leu/Ro Fro/Pro “r/c c/c 0 0

High TGF-gl production in vivo and in vitro is associated with codon 1O’Leu and codon 25*Arg. Putative ‘high, bintermediate and low producer
haplotypes are indicated.

‘Ibble 4 Frequencies of TNF-a alleles and genotypes in controls. Allele A+ (TN&) individuals produce more TNF-a upon in vitro stimulation

TNF-o position -308 polymorphism Controls (n = 106) Frequency (%)

Ahele (phenotype)
G (low) 154 73
A (high) 58 27
Genotype (phenotype)
GG (low) 65 61
GA (high) 24 23
AA (high) 17 16

‘Ihble 5 Frequencies of IL-10 alleles at position -1082 and frequency distribution of genotypes, including polymorphisms at positions -819 and
-592 in controls

IL-10 polymorphisms Controls (n = 330) Frequency (%)

Allele (phenotype)
-1082*G (high) 336 51
-1082*A (low) 324 49
-819*C 509 77
-819*T 151 23
-592*C 509 77
-592*A 151 23
Haplotypea (phenotype)
GCC (high) 336 51
ACC (intermediate) 181 27
ATA (low) 143 22
Haplotype inheritance (phenotype)
GCUGCC (high) 99 30
GCC/ACC (intermediate) 68 21
GCC/ATA (intermediate) 70 I 21
ACUACC (low) 27 8
ACC/ATA (low) 41 12
ATAIATA (low) 25 8
‘Three putative haplotypes have been identified: GCC (at positions -1082*G, -819*C and -592*C, respectively), ACC and ATA.

Transplant Immunology 1998; 6: 193-197

 Genotyping for polymorphisms: a technical report 197

%ble 6 Frequencies of IFN-7 phenotypes and frequency distribution

4 Wilson AG, di Giovine FS, Blakemore M, Duff G. Single-base poly-
of the alleles of CA repeats in controls
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#2/#5 (intermediate) 7 4.3 transplant outcome. Eur JZmmunogenet 1997; 24: 65.
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Tmnspknt Immunology 1998; 6: 193-197