Chris Perrey

Genotyping for polymorphisms in interferon-γ, interleukin-10, transforming growth factor-β1 and tumour necrosis factor-α genes: a technical report

Abstract Polymorphic variants of cytokine genes are associated with acute and chronic transplant rejection. In this technical report, the methods currently used in our centre to genotype individuals for interferon-γ, interleukin-10, transforming growth facgor-β1 and tumour necrosis factor-α 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.

CA repeat allele polymorphism in the first intron of the human interferon-gamma gene is associated with lung allograft fibrosis.

Interferon-gamma (IFN-gamma) is an inflammatory cytokine that has been implicated in the development of fibrosis in inflamed tissues. In this study we have analysed the association between genetically-determined high IFN-gamma production and development of fibrosis in lung transplants. The human IFN-gamma gene has a variable length CA repeat in the first intron. Our previous study showed that polymorphism of this microsatellite is associated with individual variation in the levels of IFN-gamma production. In vitro production of IFN-gamma showed significant correlation with presence of allele #2 (p < 0.01). In this study allele #2 was found to be associated with allograft fibrosis defined by transbronchial biopsy. An analysis of two groups of lung transplant recipients showed a significant increase in the frequency of allele #2 in the group which developed fibrosis after transplantation compared to the group that did not (p < 0.005). We postulate that the production of IFN-gamma, which is under genetic control, can influence the development of fibrosis in lung allografts.

Evidence for a genetic predisposition towards acute rejection after kidney and simultaneous kidney-pancreas transplantation.

Background. In vitro production of tumor necrosis factor-&agr; (TNF-&agr;), interferon-&ggr; (IFN-&ggr;), interleukin 10 (IL-10), and transforming growth factor-&bgr; (TGF-&bgr;) correlate with their respective genetic polymorphisms. We analyzed the relationship between these genetic polymorphisms and posttransplant outcome. Methods. Using DNA polymerase chain reaction (PCR) technology, polymorphisms for TNF- &agr;, IFN-&ggr;, IL-10, and TGF-&bgr; were determined for 82 kidney (K) and 19 simultaneous kidney-pancreas (SKP) recipients. These results were analyzed with regard to the incidence of acute rejection (AR), and the timing and severity of the first AR episode. Results. A high TNF-&agr; production phenotype correlated with recurrent acute rejection (AR) episodes (P <0.026). Compared with the low TNF-&agr; production phenotype, more patients with the high production phenotype had a post-AR serum creatinine >2.0 mg/dl, but this was not statistically significant (64 vs. 35%, P =0.12). There was no relationship between TNF-&agr; genotype and the time to first AR episode or incidence of graft loss. IFN-&ggr; production phenotype showed no correlation with any of these clinical outcome parameters. There was an increase in AR incidence as the IL-10 production phenotype increased (low, intermediate, high), but only in low TNF-&agr; producer phenotypes (P =0.023). Conclusions. Patients with a polymorphic cytokine genotype putatively encoding for high in vivo TNF-&agr; production, and to a lesser extent IL-10 cytokine genotypes putatively encoding for higher levels of in vivo IL-10 production, had a worse clinical outcome regarding AR episodes. These data support the hypothesis that the strength of alloimmune responsiveness after transplantation in part is genetically determined.

The effect of polymorphisms in tumor necrosis factor-alpha, interleukin-10, and transforming growth factor-beta1 genes in acute hepatic allograft rejection.

BACKGROUND The occurrence of acute rejection in orthotopic liver transplantation is unpredictable. The role of cytokines in the process of rejection is not entirely clear. We investigated polymorphisms in the genes encoding tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, and transforming growth factor (TGF)-beta1, which affect the amount of cytokine produced in vitro, in a liver transplant population to determine any association with acute rejection. METHOD DNA was extracted from whole blood of liver transplant patients. After amplification with polymerase chain reactions, the polymorphisms at TNF-alpha -308, IL-10 -1082, and TGF-beta1 +869 and +915 were determined using sequence-specific oligonucleotide probes. Acute cellular rejection was a clinical and histological diagnosis. RESULTS Acute cellular rejection requiring treatment occurred in 68 (48%) of 144 patients. Acute cellular rejection was significantly associated with the TNF-alpha -308 A/A genotype (P<0.02). There was no significant association with either IL-10 or TGF-beta1 polymorphisms in acute rejection. CONCLUSION Patients with a homozygous TNF-alpha -308 genotype A/A are more likely to suffer from acute cellular rejection after liver transplantation.

Cancer of the uterine cervix may be significantly associated with a gene polymorphism coding for increased IL‐10 production

The purpose of our prospective, case‐controlled study was to investigate the hypothesis that women who are genetically programmed to produce high or medium levels of IL‐10 were more likely to develop cancer of the uterine cervix than individuals genetically predisposed to low IL‐10 production. The population was recruited from patients attending gynecological clinics at 2 hospitals in Harare, Zimbabwe. Laboratory tests were performed in the Departments of Immunology, Chemical Pathology and Medical Microbiology, Medical School, University of Zimbabwe, and simultaneously at the Department of Biological Sciences, University of Manchester, United Kingdom. Included in our study were 77 women with histologically proven cancer of the uterine cervix and 69 age‐ and parity‐matched healthy women. All of the patients and healthy controls were from the Shona ethnic group that inhabits northern Zimbabwe. DNA was purified from cervical cytobrush samples obtained from women with cervical cancer. Control DNA was extracted from urine or peripheral blood samples from the healthy women. The Qiagen DNA extraction kit was used. Detection of allele A and/or G at −1082 in the promoter region of the IL‐10 gene was carried out using the ARMS‐PCR technique. Polymorphism in the amplified products was detected by gel electrophoresis in the presence of ethidium bromide and were bands visualized under UV light. The data comprise 77 women who developed invasive cervical cancer and 69 healthy women matched for age and parity. Patients with cancer were significantly (p = 0.001) more likely to be predisposed to produce higher (A/G) levels of IL‐10. The genotype encoding for high (G/G) production of IL‐10 was only observed in one cancer patient. The prevalence of low producers of IL‐10 in the cancer group was significantly lower than in the healthy women. There were no high producers amongst the healthy women. These data suggest that the genetically acquired ability to produce higher levels of IL‐10 may be a significant factor in the development of cervical cancer.

Cytokine promoter gene polymorphisms and idiopathic recurrent pregnancy loss.

Approximately one in 300 women experience recurrent pregnancy loss (RPL), the aetiology of which is unknown in at least 40% of cases. Previously, some studies have shown increased production of pro-inflammatory cytokines (tumour necrosis factor-alpha and interferon-gamma) and reduced production of anti-inflammatory cytokines (interleukin-10) by circulating blood lymphocytes isolated from these patients when compared with controls. The reasons for this are unclear. The production of these cytokines are partly under genetic control. This study investigated whether polymorphisms in these three cytokine genes known to be associated with either high or low production, are associated with idiopathic RPL. No association was found. It may be that genetic factors are not a major determinant of cytokine production during pregnancy, or alternatively it may be that the observed differences in cytokine production by peripheral lymphocytes do not accurately indicate what is occurring at the local maternofoetal interface during successful and abortive pregnancies.

Genetic variation in proinflammatory and anti-inflammatory cytokine production in multiple organ dysfunction syndrome.

ObjectivesThe objectives of this study were to examine the prevalence of genetic variation for in vitro cytokine production (tumor necrosis factor [TNF]-&agr;, interleukin-10, transforming growth factor-&bgr;1) in patients with multiple organ dysfunction syndrome, to measure circulating cytokine levels and relate these to genotype, and to identify the relationship between genetic variation and outcome. DesignProspective analysis. SettingIntensive care unit of a university teaching hospital. PatientsEighty-eight critically ill patients with multiple organ dysfunction syndrome. Measurements and Main ResultsThe frequency of the different interleukin-10 genotypes (corresponding to high, intermediate, and low interleukin-10 production in vitro) were significantly different between controls and multiple organ dysfunction syndrome patients. High interleukin-10 producers were under-represented in the multiple organ dysfunction syndrome group: This genotype occurred in 30% of controls but in only 6% of patients (p < .001). There was no relationship between interleukin-10 genotype and mortality. The frequency of TNF-&agr; genotypes was also significantly different between patients and controls. Intermediate TNF-&agr; producers were under-represented (5.7% vs. 23%) and high TNF-&agr; producers over-represented (35.2% vs. 16%) in the patient group (p < .001). TNF-&agr; genotype was not related to mortality. The distribution of TNF-&bgr; genotypes (homozygous B1, homozygous B2, and heterozygotes) was also different between controls and patients (p = .008). The B2/B2 genotype (associated with high TNF-&agr; production) tended to occur less frequently in the intensive care unit population (31% vs. 50%) and was associated with a higher mortality rate than either the B1/B1 or B1/B2 genotypes (48% vs. 11% and 33% respectively, p = .115). The combination of proinflammatory (TNF-&agr;/TNF-&bgr;) and anti-inflammatory (interleukin-10/transforming growth factor-&bgr;1) cytokine genotypes was associated with prolonged patient survival time. Patients predisposed to produce a balanced cytokine response (e.g., intermediate interleukin-10/TNF-&agr; producers) demonstrated the longest survival times, although overall mortality was no different. ConclusionA genetic predisposition to high interleukin-10 production or intermediate TNF-&agr; production in vitro may be protective of admission to the intensive care unit, although once admitted, any protection provided by these genotypes seems to be lost. TNF-&bgr; genotype conferred no advantage to patients with multiple organ dysfunction syndrome, the TNFB2 allele being associated with increased mortality. The combination of proinflammatory and anti-inflammatory cytokine genotypes supports the idea that a balanced cytokine response is favorable and was associated with prolonged patient survival time.

polymorphisms in tumour necrosis factor α, interleukin-10 and transforming growth factor β1 genes and end-stage liver disease

OBJECTIVE: To determine any relationship between polymorphisms in the genes encoding tumour necrosis factor alpha (TNFalpha), interleukin-10 (IL-10) and transforming growth factor beta1 (TGFbeta1) and end-stage liver disease. METHODS: Whole-blood samples were taken from patients attending the Scottish Liver Transplant Unit with end-stage liver disease (primary biliary cirrhosis, n = 61; alcoholic liver disease, n = 25; primary sclerosing cholangitis, n = 17; viral disease, n = 8; type 1 auto-immune hepatitis, n = 8; acute liver failure, n = 20). DNA was extracted and the polymorphisms at positions TNF -308, IL-10 -1082 and TGFbeta1 +869 and +915 were determined using sequence-specific oligonucleotide probes. Samples were also analysed from normal healthy controls. RESULTS: There was a significant difference between patients with primary sclerosing cholangitis and healthy controls, with 65% of patients (11/17) possessing at least one TNF2 allele (A at position -308) compared with 38% of controls (P = 0.02). Four of the eight patients with auto-immune hepatitis were homozygous for TNF2 while the other four were heterozygous (P = 0.001). No significant difference between controls and patients was seen in polymorphisms for IL-10 or TGFbeta1. No association between genotype and Childs class was found in primary biliary cirrhosis. CONCLUSION: Patients with primary sclerosing cholangitis and auto-immune hepatitis are more likely to possess TNF2 than normal controls. This allele has been associated with an increased production of TNFalpha in vitro and may indicate a predisposition to these inflammatory conditions.

A study of five human cytokine genes in human essential hypertension

With a view to evaluating the putative involvement of cytokine gene variants in human essential hypertension, we carried out an association (case-control) study on 174 unrelated nationals (81 hypertensives and 93 normotensives) from the Abu Dhabi Emirate (UAE), a genetically homogeneous population also characterised by the absence of traditional confounding factors such as alcohol consumption and smoking. To that end, we targeted our investigation to five candidate gene loci-transforming growth factor beta1 (TGF-beta1), interferon gamma (IFN-gamma), epidermal growth factor (EGF), interleukin-1 beta (IL-1beta) and tumour-necrosis factor (TNF-alpha) genes. We investigated the distribution of genotypes and alleles of the six following dimorphic variants: TGF-beta1(*)10(T>C) and TGF-beta1(*)25(G>C), located at codons 10 and 25, respectively, of TGF-beta1; T874A in intron 1 of IFN-gamma; G61A in exon 1 of EGF; TaqI dimorphism at +3962 (exon 5) of IL-1beta; and -308A>G in the promoter of TNF-alpha. These six bi-allelic markers were visualised by methods based on the techniques of amplification refractory mutation system-polymerase chain reaction (for TGF-beta1, IFN-gamma, EGF and TNF-alpha) and by polymerase chain reaction-TaqI restriction endonuclease analysis in the case of IL-1beta. In each of the two groups (normotensives and hypertensives), genotype frequencies of all six markers occurred in Hardy-Weinberg proportions. There were, however, no statistical differences in the allele and genotype frequencies of any of the six markers between the two groups of subjects: TGF-beta1(*)10C frequencies were 0.46 and 0.49 (chi(2)=0.61; 2 d.f.; P=0.74) and TGF-beta1(*)25C were 0.07 and 0.08 (chi(2)=0.61; 2 d.f.; P=0.74) amongst normotensives and hypertensives, respectively; p(IFN-gamma(*)A874) were 0.41 in normotensives versus 0.46 in hypertensives (chi(2)=3.07; 2 d.f.; P=0.22); p(EGF (*)G61) were 0.51 versus 0.58 (chi(2)=1.76; 2 d.f.; P=0.41); p[IL-1beta (*)TaqI(+)] were 0.43 versus 0.36 (chi(2)=2.08; 2 d.f.; P=0.35); and p(TNF-alpha(*)-308G) were 0.80 versus 0.85 (chi(2)=1.29; 2 d.f.; P=0.53). There was also no difference in distribution and frequencies of haplotypes constructed with combinations of TGF-beta1(*)10(T>C) and TGF-beta1(*)25(G>C) sites. However, although they do not reach statistical significance (which may be due to the relatively restricted number of subjects included in this study), the distribution differences (in normotensives and hypertensives) observed in the cases of EGF and TNF-alpha reflect trends that could be expected from a mechanistic explanation of the pathways that underlie the patho-physiology of hypertension.

Lack of association between human TGF-β1 gene variants and primary hypertension

Epidemiologic, clinical, and experimental evidence has suggested that upregulation of transforming growth factor-b1 (TGF-b1) may play a role in hypertensive disease. TGF-b1 is a multifunctional cytokine that regulates cell growth and differentiation, modulation of extracellular matrix and repair, which in turn regulate inflammatory and immune responses and can be involved in vascular remodelling. Among the seven polymorphisms that have been identified at the TGF-b1 gene locus, the TGF-b125C (Pro25) allele has been found to be associated with lower systolic blood pressure and lower risk of essential hypertension (EHT), but with increased risk of myocardial infarction in sample populations from Northern Ireland and France. The authors, together with an accompanying editorial, offered a cautious interpretation of their data, which therefore needs further probing in other populations. Furthermore, that study did not address the issue of quantitative phenotypes related to TGF-b1 production in relation to EHT. This approach became available through the findings that a polymorphism at position 75 in the signal peptide sequence, which changes codon 25 (Arg3Pro), is associated with interindividual variation in TGF-b1 production. The Arg-to-Pro at codon 25 is in the signal sequence of the precursor, and the mutation results in the substitution of a large polar amino acid with a small nonpolar residue. No study has so far been conducted to determine whether the substitution has functional and biologic importance, but it could affect protein transport into the endoplasmic reticulum. As data from genetically isolated populations are of utmost importance to resolve issues of contention related to studies based on the concept of linkage disequilibrium, we carried out an association study on nationals from the United Arab Emirates (UAE) with a view to evaluating the putative involvement of the TGF-b1 gene in EHT in a genetically homogeneous ethnic group. Using a method of amplification-refractory mutational system (ARMS)/polymerase chain reaction (PCR) that has been described elsewhere, we investigated in unrelated normotensive and hypertensive UAE subjects the distributions of genotypes and alleles of the two following two-allele polymorphisms: a T-to-C transition in exon 10 of the TGF-b1 gene (TGF-b110[T3C]), which leads to a Leu-to-Pro mutation; and the G-to-C transversion in exon 25 of TGF-b1 (TGF-b125[G3C]). This project was approved by the Research Ethics Committee of the Faculty of Medicine and Health Sciences (UAE University, Al Ain, UAE). The sample population of 144 unrelated subjects (75 men, 69 women) had a mean age (6 standard deviation) of 52.1 6 12.3 years and was composed of the following two groups: 72 patients with EHT (mean age, 52.8 6 12.1 years), and 72 controls used as a comparison group (mean age, 51.7 6 11.5 years). Hypertensive and normotensive subjects have been described before. They were matched for age and gender, and there was no difference in body mass index values among the two groups. Total serum cholesterol levels were significantly higher in the group of hypertensives. TGF-b110C allele frequencies were 0.46 6 0.04 and 0.41 6 0.04 among normotensives and hypertensives, respectively; TGF-b125C allele frequencies were 0.07 6 0.02 and 0.08 6 0.02 (data not shown). There was no statistically significant difference in the distributions of genotypes of either of the two individual markers according to clinical phenotype (data not shown). The overall allelic frequencies of both dimorphisms were similar to what has been found in a European population. Thus, the TGF-b110C allele frequency was 0.41 to 0.46 6 0.04 among Emirati, compared with 0.35 6 0.03 in a UK sample population and 0.41 to 0.43 in other European populations. TGFb125C allele frequencies were 0.07 to 0.08 6 0.02 among Emirati, 010 6 0.02 among UK subjects, and 0.07 to 0.11 among Irish and French subjects from different Centers. Haplotypes combining both sites were constructed and linkage disequilibrium values (gametic diallelic pairwise disequilibrium, D) for the pair of dimorphisms (10T3C and 25G3C) were measured by calculating the deviation of the observed frequency of the haplotype from that expected from multiplication of the individual allele frequencies. In the case of double heterozygotes (10T/10C with 25G/25C), the haplotype phase was estimated by the maximum-likelihood procedure described by Hill. The significance of the difference of D 5 D/Dmax from 0 (Dmax is the maximum possible disequilibrium for a given pair of allele frequencies) was calculated as x with 1 degree of freedom (df) using a method described elsewhere. The resulting values were as follows: for AJH 2000;13:944–945