# Description of three new polymorphisms in the intronic and 3' UTR regions of the human interferon gamma gene

Sandrine Henri, F Stefani, D Parzy, C Eboumbou, A Dessein, Christophe Chevillard

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Sandrine Henri, F Stefani, D Parzy, C Eboumbou, A Dessein, et al.. Description of three new polymorphisms in the intronic and 3' UTR regions of the human interferon gamma gene. Genes and Immunity, 2002, 3 (1), pp.1-4. 10.1038/sj.gene.6363809. hal-01592718

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# Description of three new polymorphisms in the intronic and 3'UTR regions of the human interferon gamma gene

S Henri¹, F Stefani¹, D Parzy², C Eboumbou¹, A Dessein¹ and C Chevillard¹

¹INSERM U399, Immunologie et Génétique des Maladies Parasitaires, Faculté de Médecine, 27 bd J. Moulin, 13385 Marseille cedex, France; ²Institut de Médecine Tropicale du Service de Santé des Armées (IMTSSA), F-13007 Marseille, France

Interferon-gamma (IFN-γ) is a key regulator of the development and functions of the immune system. In particular, this cytokine plays a major role in immune defense against infections by various human pathogens and polymorphisms in the IFN-γ gene, including the transcription regulatory region, and might affect host resistance to infectious agents such as schistosomes. In this study on the genetics of human schistosomiasis we uncovered three new single nucleotide polymorphisms in the IFN-γ genes. Two polymorphisms are located in the third intron and the third is in the 3'UTR region of this gene: an A to G transition at position +2109 from the transcription start and two G to A transitions at positions +3810 and +5134. In a SUDANESE population living in an endemic area of malaria and schistosomiasis, the allelic frequencies are: 0.85 (+2109A), 0.15 (+2109G), 0.92 (+3810G), 0.08 (+3810A), (+5134G) and 0.04 (+5134A). Genes and Immunity (2002) **3**, 1–4. DOI: 10.1038/sj/gene/6363809

**Keywords:** interferon gamma; polymorphisms; SNPs

## Introduction

Interferons are a small group of cytokines that include alpha, beta and gamma interferons (IFN-α, β, γ). IFN-γ is a pleiotropic cytokine that is a key regulator of the development and functions of the immune system. The principal sources of IFN-γ are helper T cells and natural killer cells, although many other cells can produce it. The human IFN-γ gene, consists of four exons and is located near D12S335 and D12S313 microsatellites on 12q15.¹,² It encodes a polypeptide of 166 amino acids with a 20-amino acid signal peptide.

Deletion analysis studies have defined the core IFN-γ promoter³,⁴ which covers the –100 to –30 region and is much conserved through evolution. Furthermore enhancer sites have been located in the first intron and several NFκB binding sites have been described in introns 1, 2 and 3.⁵,⁶ Genomic DNA constructs lacking most intronic sequences have consistently exhibited a decrease of transcription activity. It is not clear, however, whether NFκB/NFAT complexes have positive or negative effects on transcription. These complexes interact with CD28 and CD28 responsive elements in the promoter and in the third intron. These data indicate that

several transcription regulatory regions are distributed along the gene. We have searched for polymorphisms in the IFN-γ gene because of its important role in regulating inflammation and fibrosis in human schistosome infection. We describe here three new polymorphisms in the intronic and 3'UTR regions of the IFN-γ gene.

## Results and discussion

We searched for IFN-γ polymorphisms in SUDANESE subjects from a region of endemic schistosomiasis. We sought single-strand conformational polymorphisms (SSCP) with sets of primers covering the full gene (see primers in Table 1). Polymerase chain reaction (PCR) products were electrophoresed at 4°C and at room temperature. PCR products obtained from the DNA of patient 27 amplified with the 10a/b primers exhibited an additional band on SSCP gel compared to the products obtained from DNA of patient 33 (Figure 1a). Two other polymorphisms were detected using the 16a/b and 21a/b primer sets: PCR products obtained from the DNA of patient 38 and 26 with primers 16a/b and 21a/b respectively exhibited SSCP patterns different from other subjects (subjects 18 and 6 respectively) (Figures 2a and 3a).

Then, PCR products from subjects 27, 33 (10a/b) 18, 38 (16a/b) 6, 26 (21a/b) were sequenced in both directions. Chromatograms are shown in Figures 1b, 2b and 3b. Sequence alignments are provided on Figures 1c, 2c and 3c. Figure 1b and 1c identified an A to G transition at position +2109 from the transcription start in the third intron. Subject 33 was found to be homozygous (A/A) whereas subject 27 was heterozygous (G/T). Likewise, a G to A transition was identified at position +3810 (third intron) in Figure 2b and 2c. Subject 18 was homozygous

Correspondence: C Chevillard, INSERM U399, Immunologie et Génétique des Maladies Parasitaires, faculté de médecine, 27 bd J. Moulin, 13385 Marseille cedex, France.

E-mail: christophe.chevillard@medecine.univ-mrs.fr

This work received financial assistance from INSERM, CNRS, WHO (ID096546). C Chevillard and S Henri were supported by fellowships from the Conseil Général PACA and from the french Ministry of Research and Technologies.

Received 20 August 2001; revised and accepted 10 September 2001
<figure><img src="image_3.png" /><figcaption>**Figure 2** Analysis of the point mutation at position +3810. (a) SSCP analysis shows a polymorphism in the human interferon gamma gene. This SSCP electrophoresis was carried out at 4°C. (b) The PCR reaction products obtained from subjects 18 and 38 were sequenced with primers 16a and 16b. Chromatograms confirmed the heterozygous status of subject 38. Sequences obtained from subjects 18 and 38 have been deposited in GenBank (numbers: AF408172 and AF408173 respectively). (c) Sequence alignment located this mutation at position +3810 from the transcription start.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>**Figure 3** Analysis of the point mutation at position +5134. (a) SSCP analysis shows one polymorphism in the human interferon gamma gene. This SSCP electrophoresis was carried out at 4°C. (b) The PCR reaction products obtained from subjects 6 and 26 were sequenced with primers 21a and 21b. Chromatograms confirmed the heterozygous status of subject 26. Sequences obtained from subjects 6 and 26 have been deposited in GenBank (numbers: AF408174 and AF408175 respectively). (c) Sequence alignment located this mutation at position +5134 from the transcription start.</figcaption></figure>

patients 27 and 33 obtained with 10a/b confirmed this polymorphism. Subject 33 PCR products were not digested (one 365-bp band) whereas subject 27 PCR products were partially digested as three fragments are visible on gel (106-bp, 259-bp and 365-bp fragments) (Figure 4).

The frequency of each polymorphism were determined in the population from which originate the subjects studied above. Out of 134 subjects, 95 and 39 individuals presented the +2109A/A and +2109A/G genotype respectively. Allelic frequencies were 85% (+2109A) and 15% (+2109G). Out of 99 subjects, 84 and 15 individuals had the +3810G/G and +3810G/A genotype respectively. Allelic frequencies were 92% (+3810G) and 8% (+3810A). Finally, out of 78 subjects, 72 and six individuals had the +5134G/G and +5134G/A genotype respectively. Allelic frequencies were 96% (+5134G) and 4% (+5135A).

The frequency of each polymorphism was also determined in the CEPH reference population. In 111 individuals studied, no polymorphism was detected at positions +3810 and +5134 and all the subjects were +3810 G/G and +5134 G/G. However out of 110 subjects, 51, 48 and 11 individuals presented the +2109A/A and +2109A/G and +2109G/G genotype respectively. Allelic frequencies were 68% (+2109A) and 32% (+2109G) (Table 2).

The three new polymorphisms identified in the present study were located in the intronic and 3'UTR regions which are involved in transcriptional regulation. Although none of these new polymorphisms are located in a sequence that was previously demonstrated to be involved in gene regulation. This analysis has been performed on a SUDANESE population living in a region where *Schistosoma mansoni* infection is endemic. Lethality in *Schistosoma mansoni* infections is the consequence of portal hypertension caused by hepatic periportal fibrosis. Development of hepatic periportal fibrosis is controlled
<table><caption>**Table 2** Allelic frequencies of the three polymorphisms in the Sudanese and in the CEPH reference populations</caption><thead><tr><th rowspan="3"></th><th colspan="6">Position from the transcription start</th></tr><tr><th colspan="2">+2109</th><th colspan="2">+3810</th><th colspan="2">+5134</th></tr><tr><th>Sudanese population</th><th>CEPH population</th><th>Sudanese population</th><th>CEPH population</th><th>Sudanese population</th><th>CEPH population</th></tr></thead><tbody><tr><th colspan="7">Genotype</th></tr><tr><td>A/A</td><td>95/134 (71%)</td><td>51/110 (46%)</td><td>0/99 (0%)</td><td>0/110 (0%)</td><td>0/78 (0%)</td><td>0/100 (0%)</td></tr><tr><td>A/G</td><td>39/134 (29%)</td><td>48/110 (44%)</td><td>15/99 (15%)</td><td>0/110 (0%)</td><td>6/78 (8%)</td><td>0/110 (0%)</td></tr><tr><td>G/G</td><td>0/134 (0%)</td><td>11/110 (10%)</td><td>84/99 (85%)</td><td>110/110 (100%)</td><td>72/78 (92%)</td><td>110/110 (100%)</td></tr><tr><th colspan="7">Allelic frequencies</th></tr><tr><td>A</td><td>85</td><td>68</td><td>8</td><td>0</td><td>4</td><td>0</td></tr><tr><td>G</td><td>15</td><td>32</td><td>92</td><td>100</td><td>96</td><td>100</td></tr></tbody></table>

<figure><img src="image_5.png" /><figcaption>**Figure 4** Restriction analysis of 10a/b PCR products by AcI. The +2109 transition creates a novel Acil restriction site. The search for restriction sites was conducted using the program (http://www.firstmarket.com/cutter). 5 µl of PCR product from subjects 27 and 33 were digested by the restriction enzyme Acil (New England Biolabs, Beverly, MA, USA) under the conditions described by the manufacturer. Digested products were separated on a 2% agarose gel at 100 V.</figcaption></figure>

by a major locus on human chromosome 6 in q22-q23, closely linked to the gene coding for the α chain of the IFN-γ receptor.⁷,⁸ This localization is consistent with the strongly antifibrogenic property of IFN-γ. We are currently investigating the importance of the three novel polymorphisms described here in the immune response and in the development of fibrosis.

## Acknowledgements

We thank Dr Howard Cann for providing the CEPH reference population DNAs. We are grateful to Dr Roy Riblet for critical reading of the manuscript.

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