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, INTERLEUKIN-10, AND TRANSFORMING GROWTH FACTOR-ß1 GENES IN PATIENTS WITH CHRONIC HEPATITIS C VIRUS INFECTION
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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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promoter, interleukin-10 promoter, and transforming growth factor-ß1 leader sequence in patients with hepatitis C. Genomic DNA was obtained from 71 Egyptians and 67 Caucasians (hepatitis C and control patients). Amplification of appropriate gene segments was followed by direct sequencing. Infrequently occurring polymorphisms were identified at positions –244 and –77 of the tumor necrosis factor- promoter and at positions –851 and –657 of the interlukin-10 promoter. The G/A genotype associated with tumor necrosis factor- promoter positions –376 and –244 was more frequent in Egyptians (P =0.001 and P =0.004, respectively). The –244 G/A genotype occurred only in healthy Egyptians (P =0.024). Thus, ethnic differences in the distribution of genotypes of the tumor necrosis factor- promoter exist, which may have clinical implications on the outcome of hepatitis C. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although some ethnic differences in disease outcome may be due to a higher prevalence of HCV infection in Egyptians and African Americans, they may also be influenced by a genetic predisposition or individual host immune responses. It has been shown that cytokine genes are polymorphic, and it has been suggested that some of these mutations may influence the production of the associated cytokine and affect the host immune response in several infectious diseases. For example, in patients with chronic HCV, production of inappropriate amounts of cytokines such as tumor necrosis factor- (TNF-), interleukin-10 (IL-10), and transforming growth factor ß1 (TGF-ß1) have been associated with persistence of viremia, hepatic fibrogenesis, and even resistance to therapy with interferon-.7–10
Currently, four single base changes at positions –376, –308, –238, and –163, and a cytosine insertion at position +70, have been identified within the TNF- gene promoter (relative to the transcriptional start site).11,12 Polymorphisms at positions –238 and –308 have been shown to influence TNF- gene expression, and the polymorphism at –238 has been associated with the development of chronic active hepatitis after exposure to HCV.11,13 Four biallelic polymorphisms have also been described in the IL-10 gene promoter at positions –1082, –819, –652, and –592 (relative to the transcriptional start site) and low IL-10 production has been associated with the A allele at position -1082 in vitro.14,15 Similarly, two polymorphisms that appear to influence the levels of TGF-ß1 have been identified within the leader sequence of the gene at position +29 (codon 10) and +74 (codon 25), relative to the translational start site. The transition (T to C) at position +29, resulting in a change from leucine to proline in codon 10, may result in an alteration of the export efficiency of the newly synthesized protein, while the transition at position +74 (G to C), resulting in a change from arginine to proline in codon 25, has been associated with decreased levels of TGF-ß1 in vitro.16
Differences in genotype distribution associated with these previously reported polymorphisms in Caucasians and individuals of African origin have not been carefully evaluated to date. In this study, ethnic differences in the distribution of cytokine gene polymorphisms and associated genotypes were investigated in the TNF- and IL-10 genes, as well as the TGF-ß1 leader sequence, of both Caucasians Americans and Egyptians (North African subjects born in Egypt). We also contrast their occurrence in patients with chronic HCV infection and healthy subjects within each group.
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MATERIALS AND METHODS |
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The diagnosis of HCV infection was established by detection of antibodies to HCV using an enzyme immunoassay and strip immunoblot assay testing. The presence of HCV RNA was confirmed by a reverse transcriptase-polymerase chain reaction (RT-PCR). All Caucasians with chronic HCV infection included in this study had a liver biopsy performed. Unfortunately, liver biopsies were performed in only four Egyptians, thus excluding the possibility of an accurate comparison of histologic findings between Egyptians and Caucasians.
Isolation of DNA, PCR amplification, and sequence analyses. Retrospectively collected sera obtained from patients and controls were stored at –70°C until the time of DNA isolation. Genomic DNA was isolated from 500-L aliquots of serum using the Isoquick Nucleic Acid Extraction Kit (ORCA Research, Inc., Bothell, WA). Nucleic acid precipitation was performed in the presence of Pellet Paint co-precipitant (Novagen, Madison, WI), followed by precipitation with ethanol and resuspension of the nucleic acid pellet in 50 µL of RNase-free water. The PCR amplification and sequencing of amplification products were performed as previously described.17 All ambiguous sequencing results were reviewed, and the overall quality of the sequence data was assessed. Each sequence was determined in both directions to confirm our findings, and novel or infrequently occurring polymorphisms were confirmed by retesting using the previously described methods.17
Validation of serum results.
Liver fragments obtained by needle biopsy from four patients randomly selected from the study population were also subjected to extraction of genomic DNA using the Isoquick Nucleic Acid Extraction Kit (ORCA Research Inc.). Isolated DNA was subjected to PCR amplification of the IL-10 and TNF- promoter and the TGF-ß1 leader sequence fragments, followed by sequencing procedures as previously described for serum samples (a total of 12 polymorphic site or 30 possible genotype per each of the four patients were assessed). Sequences obtained from liver DNA and from serum DNA from the same patient were compared to validate methods.
Statistical analyses.
The SPSS Statistical Analysis Package version 8.0 (SPSS, Inc., Chicago, IL) was used for the analysis of all data. The frequencies of TNF-, IL-10, and TGF-ß1 genotypes were compared between North African and Caucasian patients by chi-square tests using Yates’ correction for small numbers. Fisher’s exact test was used for testing the independence of categorical variables. A P value 
0.05 was considered significant. Data comparing ages of patients are presented as the mean ± SD.
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RESULTS |
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gene promoter, 132 (95.6%) of 138 for the IL-10 gene promoter, and in 136 (98.5%) of 138 for TGF-ß1 leader sequence. Amplification of all three gene segments was not possible in only 2 (0.01%) of the 138 samples tested, both Egyptian blood donors. To validate our methods, the genotypes associated with the polymorphic sites of the three cytokines genes identified in sequences obtained from liver DNA were assessed for their concordance with serum results in four randomly selected patients. Complete (100%) concordance was found between the associated genotypes identified in sequences obtained from liver DNA and in those from serum DNA from the same patient.
In our analysis, we compared the distribution of the genotypes associated with the TNF-, IL-10, and TGF-ß1 polymorphisms between the Caucasian and Egyptian groups. We also compared the distribution of genotypes between blood donors and patients within both the Caucasian and Egyptian groups. The frequencies of the genotypes associated with these polymorphisms in each group are shown in Table 1
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gene promoter were identified only in Egyptian individuals. Specifically, a G to A transition at position -244 and a T to A transition at position –77 were identified within the TNF- gene promoters of this group. The presence of the G/A genotype at position -244 of the TNF- gene promoter was detected in 8 (11.9%) of 67 Egyptian specimens and none of the 66 Caucasians (P = 0.004). In fact, this transition was only detected in 8 (18.6%) of 43 Egyptian blood donors, while not found in any of the 24 Egyptian patients with chronic HCV infection (P = 0.024). The T/A genotype was identified at position –77 in only 3 (4.5%) of 67 Egyptian samples, while absent in all 66 Caucasians. However, this trend did not reach statistical significance (P = 0.082).
A statistically significant difference in the distribution of genotypes associated with the previously described polymorphism located at position –376 of the TNF- gene promoter was found between Egyptian and Caucasians. North Africans were more likely to have the G/A genotype when compared with Caucasians (13 [19.4%] of 67 versus 1 [1.5%] of 66; P = 0.001). However, there was no difference in the distribution of this genotype within the Egyptian group (9 [20.9%] of 43 blood donors versus 4 [16.7%] of 24 patients). The A/A genotype was also associated with the TNF- –376 polymorphism, but was identified in only two Egyptians, both healthy blood donors. Analysis of the North African group revealed complete concordance between the presence of a G to A transition at both positions –376 and –238. Our lack of an association between Egyptian and the G/A genotype at position –238 can be explained by the presence of five additional G/A transitions at position –238 (unassociated with any change at position –376) within the Caucasian group. Finally, no statistically significant differences in the distribution of TNF- promoter polymorphisms at positions –308 and –163 were noted either between or within the Caucasian and Egyptian groups.
We identified polymorphisms in all but one of the previously described polymorphic sites located within the IL-10 gene promoter (positions –1082, –819, and –592). In this study, the genotype G/G was the only genotype associated with position IL-10 –652. However, two infrequently occurring polymorphisms were identified at positions –851 and –657. The IL-10 -851 A/A genotype was present in 1 (1.5%) of 66 Egyptian and no Caucasians, while the G/A genotype was present in 5 (7.6%) of 66 Egyptians and 3 (4.5%) of 66 Caucasians. The IL-10 –657 genotype G/A was found to be present in only 2 (3.0%) of 66 Egyptian and 1 (1.5%) of 66 Caucasians. Interestingly, a single individual was determined to be heterozygous at both positions –851 (A/G) and –657 (A/G). Statistical analysis demonstrated no significant differences in the distribution of IL-10 genotypes either between or within the Caucasian and Egyptian groups.
The previously described polymorphisms within codons 10 and 25 of the leader sequence of the TGF-ß1 gene were also analyzed, and a similar distribution of genotypes was found in all groups. Additionally, no novel polymorphic sites were identified within the TGF-ß1 gene leader sequence in any of the individuals included in this study.
The nucleotide sequence data described here have been submitted to the GenBank nucleotide sequence database under the following accession numbers: AF247603 to AF247608.
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DISCUSSION |
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and IL-10 gene promoters and the TGF-ß1 gene leader sequence of the study subjects. We have demonstrated statistically significant differences between Egyptian and Caucasians, as well as between healthy and infected Egyptian, in the distribution of genotypes associated with polymorphisms of the TNF- gene promoter.
The TNF- gene promoter has been shown to contain numerous binding sites for transcriptional factors, suggesting that the presence of promoter polymorphisms might directly influence transcriptional regulation of the TNF- gene.11 An increasing number of studies are also implicating genetic variants of the TNF- gene in disease susceptibility, including HCV infection. In a recent study, Hohler and others found an increased frequency of the TNF- -238 A allele in patients with chronic HCV infection when compared with subjects without liver disease, suggesting that this polymorphism may increase an individuals susceptibility to chronic HCV infection.13 We were unable to confirm this association between chronic HCV infection and the TNF- -238 polymorphism within either the Egyptian or Caucasian study groups. We did, however, find a significant ethnic difference in the distribution of the G/A genotype at position –376 of the TNF- gene promoter between Caucasians and Egyptians. The G/A genotype was present at position –376 more frequently among Egyptians when compared with Caucasians and was strongly associated with the presence of a G to A transition at position –238 within the Egyptian group. While studies involving the prevalence of genotypes associated with position –376 are lacking, the results of one previous study including European subjects, in which the prevalence of the G/A genotype was 2.2%, is consistent with our findings among Caucasians.18 The strong association between the presence of the G/A genotype at positions –238 and –376 was also observed by Hamann and others.19 While position –376 is not located within any known regulatory region of the TNF- gene promoter, position –238 is located within a putative regulatory region known as the Y box. The Y box of the TNF- gene has been studied in the murine model and been linked to regulation of TNF- expression, suggesting that polymorphisms within this region could be functionally important in humans as well.20
A study by Zimmerman and others recently identified an additional polymorphic site located within the putative Y box at position –244.21 In this study, they identified a G to A transition at position –244 associated with study subjects of African origin only. Our results confirmed their findings, suggesting that there is a true ethnic difference in the distribution of genotypes within this regulatory element. In addition to this ethnic difference, the G/A genotype was identified only in of Egyptian blood donors, while completely absent in Egyptian HCV patients. Further studies will be important to fully evaluate the possible association between this G to A transition at position –244 and protection from HCV infection in the Egyptian population.
The only three individuals exhibiting the TNF- -77 T/A genotype were from the Egyptian group, suggesting that this may also represent a ethnic difference between Egyptian and Caucasians. Additional studies of this polymorphic site and its potential effects on TNF- gene transcriptional activity need to be performed.
We were unable to identify any significant differences in the IL-10 promoter polymorphisms or the leader sequence of the TGF-ß1 gene, either between or within the Egyptian and Caucasians groups, suggesting that mutations within these genes are unlikely to explain ethnic differences in the outcome of HCV infection.
In summary, we have demonstrated the existence of significant ethnic differences in the distribution of genotypes associated with the TNF- gene promoter at positions –376 and –244. We have also demonstrated a significant difference between HCV-infected individuals and healthy blood donors within the Egyptian group at position –244 of the TNF- promoter. Our results, despite the limitations of this study, suggest that inherited genetic variability may contribute, at least in part, to the differences observed between individuals as well as between Africans and Caucasians in response to HCV infection.
Received December 23, 2002. Accepted for publication March 13, 2003.
Financial support: This study was supported in part by a grant from the American Liver Foundation/Liver Scholar Award to Nizar N. Zein. Pedro G. Vidigal is a scholar of the Tropical Medicine PostGraduation Program, School of Medicine, Federal University of Minas Gerais (Belo Horizonte, Minas Gerais, Brazil), and was supported by the CAPES Foundation (Brasilia, Brazil).
Authors’ addresses: Nizar N. Zein, Division of Gastroenterology, Hepatology and Internal Medicine, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195. Jeffrey J. Germer, Division of Gastroenterology, Hepatology and Internal Medicine, Mayo Clinic and Mayo Foundation, Rochester, MN 55905. Abdel R. El-Zayadi, Cairo Liver Center, Cairo, Egypt. Pedro G. Vidigal, School of Medicine, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
Reprint requests: Nizar N. Zein, Division of Gastroenterology, Hepatology and Internal Medicine, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH, 44195, Telephone: 216-444-6126, Fax: 216-445-5477, E-mail: zeinn{at}ccf.org.
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REFERENCES |
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, and transforming growth factor-ß1 genes in chronic hepatitis C patients treated with interferon and ribavirin. J Hepatol 36: 271–277.[ISI][Medline]
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