Leigh J. Keen

Vascular endothelial growth factor gene polymorphism and acute respiratory distress syndrome.

Background: Non-cardiogenic pulmonary oedema is a characteristic feature of the acute respiratory distress syndrome (ARDS). The properties of vascular endothelial growth factor (VEGF) as a potent vascular permogen and mitogen have led to investigation of its potential role in this condition. Lower VEGF plasma levels have been linked to the presence of the T allele in the +936 CT polymorphism. We hypothesised that the presence of the T allele would be associated with the development and severity of ARDS. Methods: A cohort of 137 normal subjects, 117 ventilated patients with ARDS, and 103 “at risk” of ARDS were genotyped for the VEGF+936 CT polymorphism. The severity of physiological disturbance and mortality was determined in the ventilated cohorts. Results: The CT and TT genotype frequencies were increased in ARDS patients compared with both normal subjects (OR 2.01, 95% CI 1.13 to 3.58, p = 0.02) and those “at risk” (OR 2.05, 95% CI 1.02 to 2.20, p = 0.03). In patients with ARDS but not those “at risk”, CT and TT genotypes were associated with a higher mean APACHE III score (80.9 (4.3) v 69.3 (2.9), p<0.05). Conclusion: These data support a role for VEGF in the pathogenesis of ARDS and its associated physiological derangement.

A study of cytokine gene polymorphisms and protein secretion in renal transplantation

Although there is evidence that cytokine gene polymorphisms are associated with varying quantities of cytokine protein production, the exact role of these polymorphisms in allograft rejection remains unclear. In a previous study, we demonstrated a significant association between high IL-10 secretion in mixed lymphocyte culture (MLC), together with HLA mismatching for at least 4-6 antigens, with the occurrence of acute rejection following renal transplantation. We, therefore, wished to ascertain whether cytokine gene polymorphisms are associated with varying levels of protein secretion and/or allograft rejection in the same group of patients. Cytokine protein secretion in MLC for IL-4, IL-6, IL-10 and IFN-gamma was measured by ELISA in 49 patient-donor pairs. Protein secretion for the above cytokines was also measured in phytohaemagglutinin (PHA) stimulated cultures in 30 normal controls. In both patient and control groups, single nucleotide polymorphism analysis for IL-4 G(-590)T, IL-6 G(-174)C, IL-10 G(-1082)A, IL-10 C(-819)T, IL-10 C(-592)A, TNF-alpha G(-308)A and microsatellite analysis for IFNG (CA repeat) was performed. No correlation was found between cytokine gene polymorphisms and cytokine protein secretion in either mitogen stimulated cultures (control group) or MLC (patient group). In addition, no correlation was demonstrated between cytokine gene polymorphisms and renal allograft rejection.

The extent and analysis of cytokine and cytokine receptor gene polymorphism.

Cytokines play an important role in the regulation of normal immune function. In recent years cytokines and their receptors have been shown to be highly polymorphic. Polymorphisms in these genes have been associated with a number of immune diseases as well as organ transplant complications. The current disease association data is confusing and often contradictory. Whilst single locus analyses are the predominant form of cytokine polymorphism analysis, the use of polymorphic haplotypes is becoming increasingly common. This may help to give a clearer picture of the association of cytokine polymorphism with immune disfunction.

Haplotypes in the tumour necrosis factor region and myeloma

This study described the haplotypic structure across a region of chromosome 6 including the tumour necrosis factor (TNF) gene, and investigated its influence on the aetiology of myeloma. A total of 181 myeloma cases from the Medical Research Council Myeloma VII trial and 233 controls from the Leukaemia Research Fund Case Control Study of Adult Acute Leukaemia were included in the analysis. Genotyping by induced heteroduplex generator analysis was carried out for single nucleotide polymorphisms (SNP) located at positions −1031, −863, −857, −308 and −238 of the 5′ promoter region of TNF‐α gene, and 252 in the LT‐α gene; and five microsatellites, TNFa, b, c, d and e. Haplotypes were inferred statistically using the phase algorithm. A limited diversity of haplotypes was observed, with the majority of variation described by 12 frequent haplotypes. Detailed characterization of the haplotype did not provide greater determination of disease risk beyond that described by the TNF‐α−308 SNP. Some evidence was provided for a decreased risk of myeloma associated with the TNF‐α−308 variant allele A, odds ratio, 0·57; 95% confidence interval, 0·38–0·86. The results of this study did not support our starting hypothesis; that high producer haplotypes at the TNF locus are associated with an increased risk of developing myeloma.

Association between non-Hodgkin lymphoma and haplotypes in the TNF region

The cytokines tumour necrosis factor‐α (TNFα) and lymphotoxin‐α (LTα) are known to play key roles in B‐cell growth, differentiation and maturation. Genetic polymorphism within regulatory regions of these cytokine genes can alter expression levels and may be important in development of lymphoid malignancy. This study investigates a number of single nucleotide polymorphisms (SNPs) and microsatellite variants present within these genes in a large cohort of non‐Hodgkin lymphoma (NHL) cases including 211 cases of follicular lymphoma (FL) and 281 cases of diffuse large B‐cell lymphoma (DLBCL), and 478 unaffected controls. The study investigated whether particular alleles at these loci, or their combination across the TNF region in the form of haplotypes, may act as markers for predisposition and development of NHL. The study provided evidence for an influence of the TNF region in the susceptibility to NHL, whereby the loci −863, −857, TNFe and TNFd categorised five haplotype groups over which risk of both FL and DLBCL varied significantly. Prediction of disease risk was improved by the addition of loci to the haplotype, demonstrating the importance of considering the haplotype‐specific context of the loci in genetic risk assessment.

Relationship Between Vascular Endothelial Growth Factor + 936 Genotype and Plasma/Epithelial Lining Fluid Vascular Endothelial Growth Factor Protein Levels in Patients With and at Risk for ARDS

BACKGROUND Vascular endothelial growth factor (VEGF) is postulated to have a role in ARDS. The functional VEGF + 936 polymorphic T allele is associated with an increased susceptibility to and severity of ARDS. The reasons for this are unclear. We hypothesized that the T allele would be associated with an alteration in the relation between epithelial lining fluid (ELF) and plasma VEGF levels as a potential explanation for its association with susceptibility to and severity of ARDS. METHODS Plasma and ELF VEGF protein levels were measured by enzyme-linked immunosorbent assay from 10 at-risk patients receiving mechanical ventilation and 16 ARDS patients with the T allele, as well as 18 at-risk patients receiving mechanical ventilation and 26 ARDS patients without the T allele (wild-type CC genotype). RESULTS The T allele was associated with a significantly lower mean ELF VEGF level in ARDS patients (2,090 +/- 758 pg/mL vs 3,292 +/- 865 pg/mL, p < 0.05) and mean ELF/plasma VEGF level ratio (13.7 +/- 4.6 pg/mL vs 94.7 +/- 51.2 pg/mL, p < 0.01). There was no relation between the T allele and plasma VEGF level, oxygenation, or acute physiology score in at-risk and ARDS patients. ELF VEGF levels were significantly higher than plasma levels in both cohorts except for at-risk patients without the T allele (wild-type CC genotype). CONCLUSION The T allele is associated with a significant decrease in ELF levels and the ELF/plasma ratio in ARDS patients. This may explain the increased susceptibility and physiologic derangement in ARDS patients with the T allele. We speculate VEGF has a protective function in the lung. Further studies are necessary to clarify the underlying mechanisms.

Determination of cytokine regulatory haplotypes by induced heteroduplex analysis of DNA

Multiple single nucleotide polymorphisms (SNP) in the promoter region of the human interleukin-10 (IL-10) gene and in the signal/leader sequence of the human transforming growth factor beta 1 (TGF-beta1) gene, have been associated with susceptibility, severity and clinical outcome for a number of diseases. One common explanation for this, is that different haplotypes of these SNPs regulate the expression of the respective cytokines. Therefore, accurate determination of haplotypes by physical linkage analysis represents an important tool in investigating the pathogenesis of such diseases. Here, we demonstrate that the use of induced heteroduplex generators (IHGs) may be used to identify haplotypes within target sequences in the IL-10 and TGF-beta1 genes. Four haplotypes were observed within the IL-10 promoter region, consisting of -1082, -851, -819 and -592 SNPs. For the TGF-beta1 signal/leader sequence, we observed three haplotypes of the T869C (Leu10Pro) and G915C (Arg25Pro) SNPs. In both cases, all combinations of these haplotypes could be resolved unequivocally with a single IHG reagent.