Summary

We have detected two novel single nucleotide polymor-

phisms in the IL-2 gene, at positions –330 and +166 rel-

ative to the transcription start site. The +166 change

occurs within the leader peptide and does not affect

amino acid sequence. The –330 polymorphism has two

common alleles, making it an ideal marker for genetic

association studies.

Introduction

IL-2 is produced by activated T cells and has a power-

ful immunoregulatory effect on a variety of immune

cells (Paul & Seder, 1994). IL-2 mRNA is detected

within 30 min of T-cell stimulation. Gene transcription

appears to be dependent on continuous stimulation

(Shaw et al., 1988) and maximum steady-state levels

are achieved within 4–8 h post-exposure to stimulus.

In vitro transient transaction of the IL-2 gene pro-

moter showed that the 5' proximal 300 bp is sufficient

to mediate T-cell-specific transcription of a linked gene

(Jain et al., 1995). In this report we have examined the

region of the IL-2 gene sequence at positions –451 to

+316 for single nucleotide polymorphisms (SNPs), using

SSCP and dye terminator cycle sequencing techniques.

Two novel SNPs were detected at positions –330 and

+166 of the IL-2 gene sequence.

Materials and methods

Two PCR primers were designed to amplify the region

–451 to +316 (forward primer 5' TGA AAC AGG AAA

CCA ATA CAC T 3' and reverse primer 5' CCT GGT

GAG TTT GGG ATT C 3'). The PCR products were

digested by the FokI restriction enzyme (at 37 °C

overnight). A mixture of digested product (5 µL) and

denaturing solution (5 µL) (95% formamide, 0.1%

BPB, 0.1% Xylene cyanol) was heated for 5 min at

95 °C and chilled on ice before being applied to a 6%

polyacrylamide gel (acrylamide:bisacrylamide 29 : 1)

containing 10% glycerol. Electrophoresis was

performed at constant power (10 W) for 15 h.

Following electrophoresis, separated DNA bands were

visualized with silver staining. Samples showing

variation in banding patterns were sequenced using a

dye-primer cycle sequencing kit (Perkin Elmer).

Two single base changes were identified: at position

–330 (T→G) and position +166 (G→T). Neither poly-

morphism introduces a change in restriction enzyme

sites. The +166 polymorphism is a silent mutation not

affecting the amino acid sequence. We have established

an RFLP assay to detect the –330 polymorphism using

a new primer with a modified sequence which creates a

restriction site (C^T A G) for the enzyme MaeI. The

sequence modification was introduced to position

–333 (T→C) and the primer sequences used for this

assay were:

Forward 5' TAT TCA CAT GTT CAG TGT AGT TCT 3'

Reverse 5' TGG ATT CAC ACC CGA TTA CA 3'

PCR-RFLP was carried out as follows: 200 ng genomic

DNA was amplified in a 25-µL final volume PCR reac-

tion containing 1x buffer (KCL, Bioline), 1.5 mM

MgCl2, 0.2 mM dNTPs (Boehringer), 6.25 pmol of each

primer, 1 Unit Taq polymerase (Bioline), and 1 M

betaine. PCR was carried out on a Hybaid Omnigene

thermal cycler. The conditions were 1 cycle at 95 °C

(2 min) followed by 35 cycles each of 95 °C (1 min),

48 °C (1 min) and 72 °C (1 min). The reaction was com-

pleted by a final extension cycle of 72 °C (5 min).

Following amplification, the PCR product was visual-

ized on a 2% agarose gel stained with ethidium bro-

mide. This confirmed the presence of a 150-bp product.

MaeI enzyme digestion was carried out in a 20-µL

final volume reaction using 3 U MaeI (Boehringer-

Mannheim) and 4 µL of PCR product. The reactions

were overlaid with paraffin oil and incubated at 45 °C

© 1998 Blackwell Science Ltd, European Journal of Immunogenetics 25, 419–420

Two novel biallelic polymorphisms in the IL-2 gene

S. John, D. Turner,* R. Donn, P. Sinnott,‡ J. Worthington, W. E. R. Ollier, I. V. Hutchinson† &A. H. Hajeer

419

The nucleotide sequence data described in this paper has beensubmitted to the EMBL nucleotide sequence database and has beenassigned the accession number AJ006884.

ARC Epidemiology Research Unit, Stopford Building, Oxford Road,Manchester M13 9PT, UK, *National Blood Service, North LondonCentre, Colindale Avenue, London NW9 5BG, UK, †ImmunologyGroup, School of Biological Sciences, Stopford Building, Oxford Road,Manchester M13 9PT, UK, and ‡Tissue Typing Laboratory, The RoyalLondon Hospital, 55–76 Ashfield Street, Whitechapel, London E11BB, UK.

Received 28 July 1998; revised 7 September 1998; accepted 7September 1998

Correspondence: Dr Ali Hajeer, ARC Epidemiology Research Unit,Stopford Building, Oxford Road, Manchester M13 9PT, UK.

Short Communication

overnight. The digested products were visualized on a

4% Nuseive agarose gel (FMC) stained with ethidium

bromide. Material from individuals homozygous for the

–330T (wild type) does not cut with MaeI and remains

as a 150-bp product. The homozygous mutant (– 330G)

cuts with MaeI to give 124- and 26-bp fragments.

Frequencies for the T and G alleles were evaluated in 79

unrelated healthy Caucasians (Table 1).

Results and discussion

Two novel polymorphisms have been identified in the

IL2 gene. One is a silent mutation in the leader peptide

and therefore of no functional significance. The second

polymorphism (–330) occurs within the promoter

region and may therefore have an influence on levels of

IL2 production. In vitro studies would be required to

determine whether this polymorphism is functional.

The –330 polymorphism has two common alleles,

making it an ideal marker for association and linkage

studies in autoimmune diseases. IL2 is a strong candidate

gene in autoimmune diseases. Evidence of linkage to IL2

has been reported in both the IDDM NOD mouse model

(Denny et al., 1997) and in a subset of patients with RA

(John et al, 1998). This polymorphism provides a useful

marker to test IL2 directly in genetic studies.

ReferencesDenny, P., Lord, C.J., Hill, N.J., Goy, J.V., Levy, E.R., Podolin,

P.L., Peterson, L.B., Wicker, L.S., Todd, J.A. & Lyons, P.A.

(1997) Mapping of the IDDM locus Idd3 to a 0.35-cM interval

containing the interleukin-2 gene. Diabetes, 46, 695.

Jain, J., Loh, C. & Rao, A. (1995) Transcriptional regulation of the

IL-2 gene. Current Opinion in Immunology, 7, 333.

John, S., Myerscough, A., Marlow, A., Hajeer, A., Silman, A.,

Ollier, W. & Worthington, J. (1998) Linkage of cytokine genes

to rheumatoid arthritis. Evidence of genetic heterogeneity.Annals of Rheumatic Diseases, 57, 361.

Paul, W.E. & Seder, R.A. (1994) Lymphocyte-responses and

cytokines. Cell, 76, 241.

Shaw, J., Meerovitch, K., Bleackley, R.C. & Paetkau, V. (1988)

Mechanisms regulating the level of IL-2 messenger-RNA in

lymphocytes. Journal of Immunology, 140, 2243.

420 S. John et al.

© 1998 Blackwell Science Ltd, European Journal of Immunogenetics 25, 419–420

Table 1 Allele frequencies for IL2–330 polymorphism in 79unrelated UK Caucasoid individuals

N (%)

AlleleT 115 (72.8)G 43 (27.2)

GenotypeTT 42 (53.2)GT 31 (39.2)GG 6 (7.6)