Genetic variants of microsomal epoxide hydrolase and glutamate-cysteine ligase in COPD.

The genetic factors that contribute to the development of chronic obstructive pulmonary disease (COPD) are poorly understood. Many candidate genes have been proposed, including enzymes that protect the lung against oxidative stress, such as microsomal epoxide hydrolase (EPHX1) and glutamate-cysteine ligase (GCL). To date, most reported findings have been for EPHX1, particularly in relation to functional variants associated with fast and slow metabolism of epoxide intermediates. The present study aimed to identify any association of variation in these genes with COPD susceptibility or severity. In total, 1,017 white COPD patients and 912 nondiseased age and sex matched smoking controls were genotyped for six single nucleotide polymorphisms (SNPs) in EPHX1 (including the fast and slow variants and associated haplotypes), and eight SNPs in the two genes encoding GCL. GCL is a rate-limiting enzyme in the synthesis of glutathione, a major contributor to anti-oxidant protection in the lung. No association of variation was found in EPHX1 or GCL with susceptibility to COPD or disease severity. This is the largest reported study to date and is well powered to detect associations that have been previously suggested. The current data indicate that these genetic variants are unlikely to be related to susceptibility or disease severity in white chronic obstructive pulmonary disease patients.

Mutation in an alpha1-antitrypsin enhancer results in an interleukin-6 deficient acute-phase response due to loss of cooperativity between transcription factors.

We previously reported that a mutation in a 3' enhancer region of the alpha1-antitrypsin (AAT) gene is associated with chronic obstructive airways disease (COAD). During the acute-phase response the plasma concentration of AAT increases approximately 3-fold and this effect is mediated primarily by interleukin-6 (IL-6). We demonstrate, by transfection of Hep G2 cells, that the AAT gene contains at least two enhancers, one at the 5' end of the gene which is dominant under basal conditions, and another at the 3' end of the gene which exhibits weak basal activity. However, both enhancers must be present to modulate the IL-6-induced response which is diminished as a consequence of the 3' enhancer mutation. These results suggest that the 3' enhancer modulates the IL-6 response through an interaction with the 5' enhancer. The mutation occurs at a DNA binding site for the ubiquitous transcription factor octamer-1 (Oct-1) and results in a loss of cooperativity between Oct-1 and the tissue-specific transcription factor, NF-IL6 (C/EBPbeta), a member of the C/EBP family of transcription factors. NF-IL6 is a key transcription factor for a major pathway through which IL-6 mediates its effects. These observations provide a novel mechanism for a diminished IL-6-induced response.

Alpha 1-antichymotrypsin.

alpha 1-Antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family, inhibits neutrophil proteinase cathepsin G and mast cell chymases, and protects the lower respiratory tract from damage by proteolytic enzymes. It contains a reactive centre loop, which interacts with cognate proteinases, resulting in loop cleavage and a major conformational change. Recently, alpha 1-antichymotrypsin has been identified as a major constituent of the neurofibrillary plaques associated with Alzheimer's disease, and in vitro studies have shown that it enhances the rate of amyloid-fibril formation. These observations and recent genetic evidence suggest that alpha 1-antichymotrypsin is important in the pathogenesis of Alzheimer's disease.

Regulation of the serine proteinase inhibitor (SERPIN) gene alpha 1-antitrypsin: a paradigm for other SERPINs.

alpha 1-antitrypsin (AAT) is the archetypal member of the serine proteinase inhibitor (SERPIN) gene family. AAT is an acute-phase reactant and the plasma concentration increases three- to four-fold during the inflammatory response. In hepatocytes this increase is mediated primarily by the cytokine interleukin-6 (IL-6) via the transcription factor NF-IL6. The AAT gene contains at least two enhancer elements, one at the 5' end of the gene and the other at the 3' end. Functional studies performed in mammalian hepatoma cells (Hep G2) using constructs containing these AAT enhancer regions linked to a reporter gene have demonstrated that the 5' enhancer is dominant under basal conditions and that, following stimulation with IL-6, both enhancers are essential and the 3' enhancer plays a major role. We have identified a mutation associated with lung disease which occurs in the 3' AAT enhancer; the mutation occurs at a binding site for the ubiquitous transcription factor Oct-1. The functional significance of this mutation is a deficient IL-6 response. Using the AAT gene as a model, we describe the interactions which occur between transcription factors within the 3' enhancer and also those which take place between the 5' and 3' enhancers. These studies shed light on the molecular mechanism of the acute-phase response which could possibly be extended to other members of the SERPIN gene family.

Angiotensinogen: molecular biology, biochemistry and physiology.

Angiotensinogen is the only known substrate for the enzyme renin. Angiotensin II, the end product of the reaction, is an extremely potent vasoconstrictor and a major determinant of salt and water homeostasis. It is also a growth factor. Angiotensinogen has been identified as a non-inhibitory member of the serine proteinase inhibitor family. Although the most abundant source of plasma angiotensinogen is the liver, the use of Northern blotting and reverse transcriptase PCR techniques has confirmed angiotensinogen mRNA expression in a wide range of tissues, including the kidney, brain, vascular tissue, adrenal gland, placenta and leucocytes. The sequencing of the rat and human angiotensinogen genes has increased our understanding of this protein and its role in physiology and the pathogenesis of human disease. Early observations on the regulation of angiotensinogen are now explicable at the molecular level, with the identification of the core promoter, hormone and acute phase responsive elements and tissue-specific enhancers. The role of angiotensinogen in the aetiology of hypertensive disorders has been tested in transgenic animals, and in case-controlled genetic association and linkage studies. This review examines our current understanding of angiotensinogen, in the light of recent advances.

Evaluation of polymorphisms in the presenilin-1 gene and the butyrylcholinesterase gene as risk factors in sporadic Alzheimer's disease.

The E4 allele of the apolipoprotein E gene (APOE) is a major risk factor for late-onset Alzheimer's disease (LOAD) but is neither necessary nor sufficient to cause the disease. In this study, we investigated polymorphisms in the presenilin-1 (PS-1), and butyrylcholinesterase (BChE) genes, which have been implicated as risk factors for LOAD. Our data-set comprised 177 AD and 118 control patients, all of whom had been histopathologically confirmed following autopsy. We have tested homozygosity for the PS-1 allele 1 and possession of the BChE-K variant in association with APOE epsilon4 as risk factors in LOAD. Our findings support an association between the PS-1 polymorphism and LOAD, finding homozygosity for allele 1 associated with an approximately two-fold increased risk. Our data also show that in subjects greater than 75 years of age possession of both BChE-K and APOE-epsilon4 alleles is associated with an increased risk of LOAD, whilst the risk associated with APOE-epsilon4 allele alone is not significant.

An alpha1-antitrypsin enhancer polymorphism is a genetic modifier of pulmonary outcome in cystic fibrosis.

Lung disease is the direct cause of death in over 90% of cystic fibrosis (CF) patients. Excess neutrophil elastase is an important determinant of pulmonary disease in CF. alpha1-antitrypsin (AAT), also known as alpha1-proteinase inhibitor (alpha1PI) is a major modulator of elastase activity. We investigated the hypothesis that an enhancer polymorphism in the AAT gene would contribute to pulmonary prognosis in CF. Respiratory function, chest X-ray scores, bacterial colonisation and infective exacerbation were assessed to evaluate pulmonary disease severity in the CF group. Sixteen patients were found to have the 1237A allele, and 108 the more frequent G allele. Contrary to expectation, the patients with the 1237A allele were found to have better indices of pulmonary disease progression than those without, as indicated by less change in X-ray score (1237A: 0.2+/-0.1; 1237G: 1.2+/-0.1; P = 0.002) and fewer infective exacerbations (1237A: 2.8+/-0.6; 1237G: 4.6+/-0.3; P = 0.03) over the preceding 2 years. Also, a higher proportion of the 1237A (25%) than the 1237G (6.5%) were not colonised by Pseudomonas Aeruginosa (P = 0.04). Prospective monitoring of infections for a further 2 years confirmed a lesser propensity to infection in patients with the 1237A allele. These trends were also observed in a tightly matched sub-set of CF genotypes of similar age and sex, thus confirming that these effects were independent of the CF genotype. These results indicate that this AAT enhancer polymorphism is associated with better pulmonary prognosis in CF. Though the number of CF patients with the polymorphism is small, and these data need to be confirmed in larger studies, they suggest that a cautious approach should perhaps be taken to treatment of CF patients with supplemental AAT.

HLA class I, II & III genes in confirmed late-onset Alzheimer's disease.

We first examined all the then known alleles (1997) at the HLA-A, B, Bw, C, DRB1, 3, 4 and 5, and DQB1 loci in 55 late-onset (>65y) AD cases and 73 elderly controls from Oxford. We found an association of HLA-B7 with late-onset AD (odds ratio = 3.1, corrected P = 0.04) that was limited to apolipoprotein E epsilon4-negative subjects (odds ratio = 5.1, corrected P = 0.005). We then studied linkages with Class III genes and, finally, we sought to replicate our HLA-B7 result in cohorts from Montreal and Nottingham. Altogether, we used 299 histopathologically confirmed cases of late-onset AD and 175 controls. Our initial, clear finding was not replicated in Montreal and Nottingham, however. We also failed to support any other previously reported association of AD with an HLA gene. Though we cannot exclude distinct linkages in different cohorts as an explanation of the conflicting results of HLA/AD studies, we conclude that there is no compelling evidence of a strong, direct association between late-onset AD and any HLA Class I or II allele.

Polymorphism in oestrogen response element associated with variation in plasma angiotensinogen concentrations in healthy pregnant women.

OBJECTIVE: To investigate the hypothesis that the genotype at nucleotide A(-20)C in the 5' flanking region of the angiotensinogen gene, which lies within a sequence with high homology to an oestrogen response element, affects plasma angiotensinogen levels in pregnancy. DESIGN: Prospective observational study METHODS: Seventy-two healthy pregnant women were recruited in the second half of pregnancy from hospital and primary care antenatal clinics in Nottingham, UK. Plasma angiotensinogen concentrations were measured by radioimmunoassay of angiotensin I generated from endogenous angiotensinogen in the presence of excess human renin. DNA was extracted from peripheral venous blood, and angiotensinogen genotype determined at A(-20)C, G(-6)A and Met235Thr. Associations between genotype and plasma angiotensinogen concentration were assessed by analysis of variance. RESULTS: Women homozygous for the -20C allele had the lowest mean plasma angiotensinogen concentration of 1.7 +/- 0.3micromol/l. Women homozygous for -20A had significantly higher plasma angiotensinogen concentrations (2.6 +/- 0.1 micromol/l), and intermediate levels (2.0 +/- 0.1 micromol/l) were observed in women heterozygous for A(-20)C (P = 0.002, ANOVA). The polymorphisms at nucleotide -6 and codon 235 were in almost complete linkage disequilibrium, and nucleotide -20C was found only in a subset of -6A/235Thr alleles. Conclusion The low plasma angiotensinogen levels associated with the -20C/-6A/235Thr haplotype in pregnant women contrast with the high concentrations associated with the 235Thr allele in the non-pregnant state. A possible explanation lies in the presence of a motif with high homology to an oestrogen response element between the TATA box and transcription initiation site. Previous in vitro studies of reporter gene constructs have demonstrated thatthe A(-20)C polymorphism affects oestrogen responsiveness. The results of this study support the hypothesis that the oestrogen response element of the angiotensinogen gene is of functional importance in pregnancy, and that oestrogen responsiveness in pregnancy is influenced by the genotype at nucleotide - 20.

Angiotensin-converting enzyme insertion-deletion polymorphism in normotensive and pre-eclamptic pregnancies.

OBJECTIVE: To investigate the hypothesis that pre-eclampsia is associated with a common insertion-deletion polymorphism in the angiotensin-converting enzyme gene. DESIGN: Seventy-two women with pre-eclampsia and 83 normotensive pregnant women participated in the study. Pre-eclampsia was defined as a blood pressure exceeding 140/90 mm Hg in a previously normotensive woman, associated with proteinuria in excess of 300 mg/l in a 24 h collection. Samples for fetal genotyping were available from 66 pregnancies complicated by pre-eclampsia and 79 normotensive pregnancies. METHODS: Maternal and fetal samples were genotyped at the insertion-deletion (I-D) polymorphism in intron 16 of the angiotensin-converting enzyme gene by the polymerase chain reaction followed by agarose electrophoresis. RESULTS: Neither the I-D genotype distributions nor the allele frequencies differed significantly between pre-eclamptic and normotensive pregnancies in maternal or fetal samples (phi2 <0.3, not significant). The odds ratio for pre-eclampsia in women with the DD genotype, compared with the ID and II genotype, was 1.09 (95% confidence interval 0.55-2.16). The odds ratio associated with the DD genotype in the fetus was 1.14 (0.56-2.32). CONCLUSION: This study has found no evidence that the insertion-deletion polymorphism in the angiotensin-converting enzyme gene is associated with pre-eclampsia.

Functional and genetic studies of the angiotensin II type 1 receptor in pre-eclamptic and normotensive pregnant women.

OBJECTIVE: To examine and compare angiotensin II type 1 receptor genotype and its relationship to platelet angiotensin II binding for pre-eclamptic and normotensive pregnant women. DESIGN: In a case-control study, 43 pre-eclamptic women and 83 normotensive women were genotyped at the angiotensin II type 1 receptor gene locus. Platelet angiotensin II binding was measured for a subset of 11 pre-eclamptic and 57 normotensive pregnant women. We genotyped 162 healthy blood donors also, to examine the allelic background and patterns of linkage disequilibrium in the Nottingham population. METHODS: Patients were recruited during pregnancy using a rigorous definition of pre-eclampsia. DNA was extracted from peripheral venous blood and genotyped at six previously described diallelic polymorphisms in the angiotensin II type 1 receptor gene, using competitive allele-specific oligonucleotide hybridization, and at a dinucleotide repeat polymorphism in the 3' flanking region of the gene. Platelet angiotensin II binding and plasma angiotensin II concentrations were determined for peripheral venous blood. RESULTS: Normotensive pregnant women homozygous for cytosine at nucleotide 573 had significantly higher levels of platelet angiotensin II binding than did heterozygous women and women homozygous for thymidine at this site. Pre-eclamptic women had significantly higher levels of platelet angiotensin II binding than did normotensive pregnant women. The frequencies of allelic variants did not differ significantly between normotensive and pre-eclamptic women. CONCLUSION: The physiological regulation of platelet angiotensin II type 1 receptor expression in normal pregnancy is determined in part by angiotensin II type 1 receptor genotype. There was no evidence that the polymorphisms in the angiotensin II type 1 receptor gene were associated with pre-eclampsia.

Independent mutations in the flanking sequence of the alpha-1-antitrypsin gene are associated with chronic obstructive airways disease.

Two restriction fragment length polymorphisms (RFLPs) of the flanking sequence of the alpha 1-antitrypsin (AAT) gene, detected with the restriction enzymes HindIII and TaqI, have been reported to occur more commonly in patients with chronic obstructive airways disease (COAD). Their frequencies were investigated in 20 Caucasian families with a family history of COAD and in 140 unrelated COAD patients none of whom had AAT deficiency. The HindIII polymorphism was present in six index cases of 20 families (p = 0.0015) and 14 of the unrelated patients (p = 0.061) compared with one of 60 healthy unrelated controls. The TaqI polymorphism was present in five of 101 healthy unrelated controls and in three index cases of the 20 families. In the unrelated patient group 28 of 140 had the polymorphism (chi 2 = 10.01, p = 0.0016) and corresponds to a mean log odds ratio of 1.56 (95 per cent confidence limits of 0.58-2.56). The polymorphisms occurred independently of each other and were not associated with AAT deficiency in the basal state.

A polymorphism in the tumor necrosis factor-alpha gene promoter region may predispose to a poor prognosis in COPD.

STUDY OBJECTIVES: To determine whether the adenine (A)-guanine (G) substitution polymorphism at position - 308 on the tumor necrosis factor-alpha gene confers susceptibility to COPD or to the development of a more severe form of disease. DESIGN: A cross-sectional study was undertaken to compare the frequency of the A allele in a group of 106 patients with COPD with that in a control population (n = 99). Patients were followed up prospectively for a period of 2 years. PARTICIPANTS AND SETTING: Participants included 106 COPD patients recruited from a respiratory outpatient clinic and 99 control subjects recruited from patients admitted for cardiac catheterization. MEASUREMENTS AND RESULTS: DNA was extracted from venous blood, and each subject was genotyped for the polymorphism by polymerase chain reaction amplification and restriction digestion using Nco1. There was no increased frequency of the A allele in patients compared to control subjects. AA homozygous patients had less reversible airflow obstruction (p<0.05) and a significantly greater mortality (both all-cause and respiratory deaths) on follow-up (p<0.001), despite a shorter cigarette smoking history. CONCLUSIONS: This study suggests that homozygosity for this A allele predisposes to more severe airflow obstruction and a worse prognosis in COPD.

Molecular pathology of alpha 1-antitrypsin deficiency and its significance to clinical medicine.

alpha 1-Antitrypsin (AAT) deficiency is associated with predisposition to developing liver cirrhosis in early childhood, and chronic degenerative lung disease in early adult life. The probable molecular basis for the disease associations is known. One of the common variants, Z, has the propensity to form polymers, a phenomenon which is concentration- and temperature-dependent. This results in accumulation of the protein in hepatocytes, the predominant tissue source of AAT, and leads to cell damage. AAT deficiency results in loss of protection in the lung against neutrophil elastase (NE) the major target for AAT. NE is capable of destroying the architecture of the lung, leading to pulmonary emphysema. The disease process is exacerbated by cigarette smoke, which is capable of oxidizing a critical methionine residue at the active site, rendering AAT an inefficient inhibitor of NE. The combination of deficiency and cigarette smoking are critical to the development of pulmonary emphysema. We have identified a mutation in an enhancer sequence which, in all probability, predisposes to disease by a novel mechanism related to diminished expression of AAT during inflammation. Our understanding of the mechanisms of disease should lead to improved therapeutic interventions.

Alpha 1-antitrypsin: structure, function and molecular biology of the gene.

Alpha 1-antitrypsin (AAT) deficiency is one of the commonest inherited disorders in white Caucasians. This association has provided major insights into the pathogenesis of chronic lung disease. The three dimensional structure of the protein and the structure of the gene have been determined. Some of the signals required for regulation of expression and tissue-specificity have been defined. Genetic manipulation of active site residues may provide a new generation of biological compounds with potential therapeutic applications.

Possible applications of recombinant DNA technology to the diagnosis and study of variants of alpha 1-antitrypsin.

We describe the basic principles of recombinant DNA technology and how this methodology has been used to isolate and characterise cloned genes coding for alpha 1-antitrypsin. We show how these probes can be used for diagnosis and to study molecular variants of alpha 1-antitrypsin which may predispose individuals to develop lung disease.

Serine proteinase inhibitors on chromosome 14 and the genetics of familial chronic obstructive airways disease.

Environmental and genetic factors contribute to familial chronic obstructive airways disease. The genetic component could be polygenic or in some families be associated with one or two major genes. It is assumed that most cases of familial chronic obstructive airways disease are polygenic, but this conclusion is based on insufficient data. The use of linkage analysis using DNA probes for specific genes that may have a direct role in the disease process should facilitate our understanding of the genetics. Genetic deficiency of alpha1-antitrypsin is associated with predisposition to pulmonary emphysema. In the absence of alpha 1-antitrypsin deficiency I suggest that a study of serine-proteinase inhibitors on chromosome 14 may identify a significant proportion of families where only one major gene is important.

A patient with the rare alpha-1-antitrypsin variant (Z)bristol in compound heterozygosity with the Z mutation.

Alpha-1-antitrypsin (AAT) is a protease inhibitor (PI), deficiency of which is associated with emphysema and liver disease. The most common deficiency alleles are the S (p.Glu288Val) and Z (p.Glu366Lys) alleles. The Z allele predisposes the AAT protein to polymerization with accumulation in hepatocytes leading to liver disease in PIZ individuals. Most AAT variants have a characteristic pattern of isoforms by isoelectric focusing (IEF). A novel AAT variant called PIZbristol (p.Thr109Met) with an unusual pattern on IEF was described in 1997. We report a patient with the PIZZbristol phenotype that has not been previously described. A 43-year-old man was referred by his GP to a respiratory clinic for breathlessness. His AAT concentration was 0.50 g/L (reference range 1.0-2.0 g/L). An unusual pattern on IEF was seen and sequencing revealed the presence of the rare variant Zbristol in combination with the Z mutation. This is the second reported case of Zbristol and the first in combination with the Z mutation. The patient maintained plasma AAT concentrations around 0.50-0.70 g/L which suggested that the Zbristol protein contributed to the low plasma concentration of AAT. The clinical symptoms associated with PIZ are usually attributed to the plasma deficiency, but his only respiratory complaint was that of breathlessness. This suggests that the PIZZbristol phenotype may confer an effect on respiratory function but is not involved in liver disease.