Paul J. Sinnott

Genotypic variation in the transforming growth factor-β1 gene : Association with transforming growth factor-β1 production, fibrotic lung disease, and graft fibrosis after lung transplantation

BACKGROUND Transforming growth factor (TGF)-beta1 is a profibrogenetic cytokine that has been implicated in the development of fibrosis in transplanted tissues. In this study, we have analyzed the genetic regulation of TGF-beta1 production in lung transplant recipients. METHOD A polymerase chain reaction-single-stranded conformational polymorphism technique was used to detect polymorphisms in the TGF-beta1 gene from genomic DNA. Polymorphisms were shown to correlate with in vitro TGF-beta1 production by stimulated lymphocytes. A single-specific oligonucleotide probe hybridization method was devised to screen for these polymorphisms in lung transplant groups and controls. RESULTS We have identified five polymorphisms in the TGF-beta1 gene: two in the promoter region at positions -800 and -509, one at position +72 in a nontranslated region, and two in the signal sequence at positions +869 and +915. The polymorphism at position +915 in the signal sequence, which changes codon 25 (arginine-->proline), is associated with interindividual variation in levels of TGF-beta1 production. Stimulated lymphocytes of homozygous genotype (arginine/arginine) from control individuals produced significantly more TGF-beta1 in vitro (10037+/-745 pg/ml) compared with heterozygous (arginine/proline) individuals (6729+/-883 pg/ml; P<0.02). In patients requiring lung transplantation for a fibrotic lung condition, there was an increase in the frequency of the high-producer TGF-beta1 allele (arginine). This allele was significantly associated with pretransplant fibrotic pathology (P<0.02) (n=45) when compared with controls (n=107) and with pretransplant nonfibrotic pathology (P<0.004) (n=50). This allele was also associated with allograft fibrosis in transbronchial biopsies when compared with controls (P<0.03) and with nonallograft fibrosis (P<0.01). CONCLUSION The production of TGF-beta1 is under genetic control, and this in turn influences the development of lung fibrosis. Hence, the TGF-beta1 genotype has prognostic significance in transplant recipients.

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.

Cytokine gene polymorphism and heart transplant rejection

BACKGROUND Allograft rejection is mediated by cytokines. As polymorphism in cytokine genes can result in interindividual differences in cytokine production, we hypothesize that some patients may have an increased risk of rejection. METHODS We have related polymorphisms that influence cytokine production in the tumor necrosis factor (TNF)-A and interleukin (IL)-10 genes to early graft rejection in 115 heart transplant recipients. RESULTS Certain combinations of TNF-A and IL-10 gene polymorphisms are associated with rejection. Five of 19 patients who had high levels of rejection typed as high TNF-alpha/low IL-10 producers compared with 4 of 96 patients with lower levels of rejection (P<0.005). CONCLUSIONS We have identified a particular cytokine genotype that may confer susceptibility to increased levels of early rejection. Patients with a worse prognosis may be able to be identified pretransplant by DNA analysis of TNF-A, IL-10, and other gene polymorphisms.

IL-10 gene promoter polymorphisms in rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine which may modulate disease expression in RA. Three dimorphic polymorphisms within the IL-10 gene promoter have recently been identified and appear to influence regulation of its expression. The 1082*A allele has been associated with low and the 1082*G allele with high in vitro IL-10 production. We have analysed 117 unrelated Caucasoid RA patients and 119 ethnically matched controls. No significant differences in the allele frequencies of the three polymorphisms were found between controls and RA patients. In contrast, a significant association between the 1082*A allele and the (-1082*A/-819*C/-592*C) haplotype and IgA RF+ve/IgG RF-ve patients was observed. The association of genotypes encoding low IL-10 production with IgA RF in RA is incompatible with its suggested role in antibody isotype switching. IgA RF has been associated with severe RA and may thus be indirectly correlated with a genotype encoding low IL-10 production.

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.

Association of polymorphisms in the human interferon-gamma and interleukin-10 gene with acute and chronic kidney transplant outcome: the cytokine effect on transplantation.

Background. Our group has previously described five different size alleles of an interferon (IFN)-&ggr; microsatellite. Analyzing this polymorphism, this study correlated high IFN-&ggr; production with a 12 CA repeat allele (allele 2). Further, our group has described interleukin (IL)-10 polymorphism defining in vitro high and low IL-10 producer status. Methods. Samples from 88 of 115 consecutive cadaveric renal transplants were used to define polymorphism of both IFN-&ggr; and IL-10. Patients were separated into high and low genotypes based on the previously reported association between certain genotypes and in vitro production. Graft survival, acute rejection, and serum creatinine at 5 years were analyzed for comparison between groups. Results. The genotype associated with high IFN-&ggr; production was found in 70 patients. The incidence of acute rejection was 54.3% in the high IFN-&ggr; genotype group, compared with 44.4% in the low IFN-&ggr; group. Requirement for antithymocyte globulin therapy was greater in the high IFN-&ggr; group (odds ratio [OR]=2.5). Among HLA-DR-mismatched patients, IFN-&ggr; genotype was more strongly associated with rejection (OR=2.86). In the cyclosporine monotherapy subgroup, patients with high IFN-&ggr; genotype had a 61% incidence of rejection compared with only 20% in the low IFN-&ggr; genotype patients (OR=3.06). Graft survival was similar between the two groups. When the analysis was controlled for the presence of delayed graft function, 40.5% of the high IFN-&ggr; genotype patients had serum creatinine levels above 200 &mgr;mol/L compared with only 14.3% of the low IFN-&ggr; genotype recipients at 5 years after transplantation (P =0.05). The high IL-10 genotype was shown to be associated with better graft function at 5 years (75 vs. 50%, P =0.09). Conclusion. In this study we have shown that high producer genotype for IFN-&ggr; may have an influence on acute rejection of kidney transplants, particularly in patients on cyclosporine monotherapy. It is also associated with worse long-term graft function. On the contrary high IL-10 production may have a long-term protective effect.

Pretransplant Donor-Specific Antibodies in Cytotoxic Negative Crossmatch Kidney Transplants: Are They Relevant?

Background. The corresponding antigens of alloantibodies identified in patients awaiting kidney transplantation are often listed as unacceptable for transplantation. The use of solid phase testing, being more sensitive and accurate than conventional complement-dependent cytotoxicity (CDC) assays, has resulted in increased identification of alloantibodies. We aimed to study the clinical importance of alloantibodies defined solely by solid phase techniques. Methods. All patients transplanted between 1999 and 2001 at our center with available day-of-transplant sera (D0) were included (121 patients). All had negative CDC crossmatches. Results. Thirty-eight patients (31%) had detectable alloantibodies using high-definition assays with 16 having donor-specific antibodies (DSA) and 22 non-DSA. There were no cases of hyperacute rejection in any of the groups. Biopsy-proven acute rejection rates in the DSA and non-DSA were similar to the unsensitized group. Delayed graft function and 1-year graft survival rates were also similar for the three groups as were median 1-year serum creatinine levels. Multivariate analysis, however, showed that DSA were associated with an increased relative risk of longer-term graft failure (relative risk, 6.5; P<0.05). Conclusions. These data show that in the context of a CDC-negative crossmatch, the presence of D0 DSA has little impact on any early graft parameters. DSA, however, are associated with poorer longer-term graft outcomes in kidney transplantation.

Transforming growth factor-beta (TGF-β1) genotype and lung allograft fibrosis

BACKGROUND TGF-beta1 is a prosclerotic cytokine implicated in fibrotic processes. Fibrosis of the pulmonary parenchyma and airways is a frequent presentation in lung transplant recipients before and after transplantation. There are two genetic polymorphisms in the DNA sequence encoding the leader sequence of the TGF-beta1 protein, located at codon 10 (either leucine or proline) and at codon 25 (either arginine or proline). The codon 25 arginine allele is associated with higher TGF-beta1 production by cells activated in vitro. We tested the hypothesis that inheritance of alleles of the TGF-beta1 gene conferring higher production of TGF-beta1 may be responsible for over-expression of TGF-beta1 in transplant recipients resulting in lung allograft fibrosis. METHODS We extracted DNA from leukocytes collected from 91 pulmonary transplants performed at our centre and 96 normal healthy volunteers between May 1990 and September 1995. Part of the first exon was amplified by PCR. Samples were genotyped by using sequence specific oligonucleotide probes. RESULT The distribution of codon 10 alleles was similar in a normal healthy control group and in lung transplant recipients, regardless of their pretransplant lung pathology. By contrast, there was a significant difference in the frequency of codon 25 alleles between the control and transplant groups. In the normal control group 81% were codon 25 arginine/arginine (A/A) homozygotes, 19% were arginine/proline (A/P) heterozygotes and none were proline/proline (P/P) homozygotes. The distribution of codon 25 alleles was similar in lung transplant recipients who did not have a significant fibrosis in pretransplant pathology, but in transplant recipients who came to transplantation with lung fibrosis 98% (41 of 42 patients) were homozygous for the codon 25 A/A allele (p < .05). After lung transplantation 39 of 91 patients developed lung allograft fibrosis, and of these 92.3% (36 of 39 recipients) were of homozygous codon 25 A/A high TGF-beta1 producer genotype (p < .001). Lung transplant recipients who were homozygous for both codon 10 L/L and codon 25 A/A showed poor survival compared with all other TGF-beta1 genotypes (p < .03). CONCLUSION Homozygosity for arginine at codon 25 of the leader sequence of TGF-beta1 that correlates with higher TGF-b production in vitro, is associated with fibrotic lung pathology before lung transplantation and with the development of fibrosis in the graft. In combination with the codon 10 leucine allele, homozygosity for the codon 25arginine allele is a marker for poor post-transplant prognosis and recipient survival.