Autoantibodies of IgM and IgG classes show differences in recognition of multiple autoantigens in chronic obstructive pulmonary disease.

Autoimmunity occurs in chronic obstructive pulmonary disease (COPD). We describe an antigen microarray for detecting serum autoantibodies (AAbs) to determine how IgM, as well as IgG, AAbs distinguish patients with COPD from controls with a history of smoking without COPD. All COPD patients' sera contained elevated levels of AAbs to some of 30 autoantigens. There were significant differences in the autoantigenic specificities of IgM AAbs compared to IgG AAbs in the COPD sera: for example, AAbs to histone and scl-70 were mainly IgG, whereas AAbs to CENP-B and La/ssB were mainly IgM; by contrast, IgM and IgG AAbs to collagen-V were equally prevalent. Thus, a combination of IgM and IgG AAbs specific for multiple autoantigens are detected in all cases of COPD at a level at which all non-COPD controls are negative for AAbs. This highlights the importance of different classes of AAbs to a range of autoantigens in COPD.

Exome-wide analysis of rare coding variation identifies novel associations with COPD and airflow limitation in MOCS3, IFIT3 and SERPINA12.

BACKGROUND: Several regions of the genome have shown to be associated with COPD in genome-wide association studies of common variants. OBJECTIVE: To determine rare and potentially functional single nucleotide polymorphisms (SNPs) associated with the risk of COPD and severity of airflow limitation. METHODS: 3226 current or former smokers of European ancestry with lung function measures indicative of Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2 COPD or worse were genotyped using an exome array. An analysis of risk of COPD was carried out using ever smoking controls (n=4784). Associations with %predicted FEV1 were tested in cases. We followed-up signals of interest (p<10(-5)) in independent samples from a subset of the UK Biobank population and also undertook a more powerful discovery study by meta-analysing the exome array data and UK Biobank data for variants represented on both arrays. RESULTS: Among the associated variants were two in regions previously unreported for COPD; a low frequency non-synonymous SNP in MOCS3 (rs7269297, pdiscovery=3.08×10(-6), preplication=0.019) and a rare SNP in IFIT3, which emerged in the meta-analysis (rs140549288, pmeta=8.56×10(-6)). In the meta-analysis of % predicted FEV1 in cases, the strongest association was shown for a splice variant in a previously unreported region, SERPINA12 (rs140198372, pmeta=5.72×10(-6)). We also confirmed previously reported associations with COPD risk at MMP12, HHIP, GPR126 and CHRNA5. No associations in novel regions reached a stringent exome-wide significance threshold (p<3.7×10(-7)). CONCLUSIONS: This study identified several associations with the risk of COPD and severity of airflow limitation, including novel regions MOCS3, IFIT3 and SERPINA12, which warrant further study.

PCR Analysis to Identify AAT Gene Promoters and Splice Variants.

This chapter explores the methods used for the analysis of alpha1-antitrypsin gene expression. This includes the use of the polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), and whole transcriptome analysis combined with parallel DNA sequencing to understand the processes involved in AAT expression.

Matrix metalloproteinase-12 (MMP-12) SNP affects MMP activity, lung macrophage infiltration and protects against emphysema in COPD.

BACKGROUND: Recent genetic and animal studies have implicated matrix metalloproteinase-12 (MMP-12) in the pathogenesis of chronic obstructive pulmonary disease (COPD). It has previously been shown that individuals homozygous for the A/A allele of rs652438 in MMP-12 are over-represented among patients with severe COPD (n=1517). A study was undertaken to examine the functional basis of these findings. METHODS: rs652438 A and G variants were generated by site-directed mutagenesis and transfected into COS7 cells where they were expressed. Casein zymography and a specific FRET activity assay were used to compare MMP-12 activity between alleles. Cell migration was examined using a transwell assay. Patients from two COPD cohorts were genotyped for rs652438 and associated with inflammatory cell number in bronchoalveolar lavage fluid (n=10) and induced sputum (n=262); the emphysema score (n=1428) was assessed by CT scanning. RESULTS: Mean MMP activity was 2.95-fold higher by zymography (p=0.0049) and 3.45-fold higher by FRET assay (p=0.0001) for the A allele than the G allele. Mean migration of COS7 cells expressing the A allele was 2.31-fold greater than for those expressing the G allele (p=0.0001). Macrophage numbers were greater in bronchoalveolar lavage fluid (1.28-fold increase, p=0.033) and induced sputum (1.58-fold increase, p=0.083) of A/A individuals compared with A/G heterozygotes. The presence of the A allele was dose-dependently associated with increased emphysema (p=0.016). CONCLUSIONS: The rs652438 SNP alters MMP-12 activity with the A allele being more active, which is associated with increased macrophage infiltration and emphysema in the lungs of patients with COPD. These findings further implicate MMP-12 and this SNP in COPD.

The role of IREB2 and transforming growth factor beta-1 genetic variants in COPD: a replication case-control study.

BACKGROUND: Genetic factors are known to contribute to COPD susceptibility and these factors are not fully understood. Conflicting results have been reported for many genetic studies of candidate genes based on their role in the disease. Genome-wide association studies in combination with expression profiling have identified a number of new candidates including IREB2. A meta-analysis has implicated transforming growth factor beta-1 (TGFbeta1) as a contributor to disease susceptibility. METHODS: We have examined previously reported associations in both genes in a collection of 1017 white COPD patients and 912 non-diseased smoking controls. Genotype information was obtained for seven SNPs in the IREB2 gene, and for four SNPs in the TGFbeta1 gene. Allele and genotype frequencies were compared between COPD cases and controls, and odds ratios were calculated. The analysis was adjusted for age, sex, smoking and centre, including interactions of age, sex and smoking with centre. RESULTS: Our data replicate the association of IREB2 SNPs in association with COPD for SNP rs2568494, rs2656069 and rs12593229 with respective adjusted p-values of 0.0018, 0.0039 and 0.0053. No significant associations were identified for TGFbeta1. CONCLUSIONS: These studies have therefore confirmed that the IREB2 locus is a contributor to COPD susceptibility and suggests a new pathway in COPD pathogenesis invoking iron homeostasis.

A novel monoclonal antibody to characterize pathogenic polymers in liver disease associated with alpha1-antitrypsin deficiency.

UNLABELLED: Alpha(1)-antitrypsin is the most abundant circulating protease inhibitor. The severe Z deficiency allele (Glu342Lys) causes the protein to undergo a conformational transition and form ordered polymers that are retained within hepatocytes. This causes neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. We have developed a conformation-specific monoclonal antibody (2C1) that recognizes the pathological polymers formed by alpha(1)-antitrypsin. This antibody was used to characterize the Z variant and a novel shutter domain mutant (His334Asp; alpha(1)-antitrypsin King's) identified in a 6-week-old boy who presented with prolonged jaundice. His334Asp alpha(1)-antitrypsin rapidly forms polymers that accumulate within the endoplasmic reticulum and show delayed secretion when compared to the wild-type M alpha(1)-antitrypsin. The 2C1 antibody recognizes polymers formed by Z and His334Asp alpha(1)-antitrypsin despite the mutations directing their effects on different parts of the protein. This antibody also recognized polymers formed by the Siiyama (Ser53Phe) and Brescia (Gly225Arg) mutants, which also mediate their effects on the shutter region of alpha(1)-antitrypsin. CONCLUSION: Z and shutter domain mutants of alpha(1)-antitrypsin form polymers with a shared epitope and so are likely to have a similar structure.

Deletion of Serpina1a, a murine α1-antitrypsin ortholog, results in embryonic lethality.

Chronic obstructive pulmonary disease (COPD) is the fourth leading cause of death in the United States Approximately 1% to 2% of COPD patients suffer from α(1)-antitrypsin (A1AT) deficiency, the major inheritable predisposition to COPD/emphysema. To further study the role of A1AT deficiency in the pathogenesis of COPD/emphysema, the authors attempted to generate null-mutant mice for Serpina1a, 1 of 2 A1AT orthologs in mice. Here the authors show that targeted deletion of Serpina1a results in embryonic lethality prior to 8.5 days post conception (dpc). The results are surprising given that A1AT-null humans exist and therefore do not require this gene product for normal development. The Serpina1 gene cluster is substantially different between mouse and man. Through gene duplication, mice have 3 to 5 (depending on the strain) highly homologous proteinase inhibiting (Pi) genes, 2 of which inhibit neutrophil elastase. Despite the abundance of Pi genes in mice, Serpina1a serves a critical, nonredundant function during early mouse development. A1AT-deficient mice have been highly sought after to study emphysema, cancer, and liver disease, and as a model to perfect gene replacement therapy. These results highlight important differences between human and murine serpins and point to the difficulty inherent to using gene-targeted mice to study this common human genetic disease.

EDNRA variants associate with smooth muscle mRNA levels, cell proliferation rates, and cystic fibrosis pulmonary disease severity.

Airway inflammation and pulmonary disease are heterogeneous phenotypes in cystic fibrosis (CF) patients, even among patients with the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Endothelin, a proinflammatory peptide and smooth muscle agonist, is increased in CF airways, potentially contributing to the pulmonary phenotype. Four cohorts of CF patients were screened for variants in endothelin pathway genes to determine whether any of these variants associated with pulmonary function. An initial cohort of 808 CF patients homozygous for the common CF mutation, DeltaF508, showed significant association for polymorphisms in the endothelin receptor A gene, EDNRA (P = 0.04), but not in the related endothelin genes (EDN1, EDN2, EDN3, or EDNRB) or NOS1, NOS2A, or NOS3. Variants within EDNRA were examined in three additional cohorts of CF patients, 238 patients from Seattle, WA, 303 from Ireland and the U.K., and 228 from Cleveland, OH, for a total of 1,577 CF patients. The three additional groups each demonstrated a significant association between EDNRA 3'-untranslated region (UTR) variant rs5335 and pulmonary function (P = 0.002). At the molecular level, single nucleotide primer extension assays suggest that the effect of the variants is quantitative. EDNRA mRNA levels from cultured primary tracheal smooth muscle cells are greater for the allele that appears to be deleterious to lung function than for the protective allele, suggesting a mechanism by which increased receptor function is harmful to the CF airway. Finally, cell proliferation studies using human airway smooth muscle cells demonstrated that cells homozygous for the deleterious allele proliferate at a faster rate than those homozygous for the protective allele.

Association of MMP-2 polymorphisms with severe and very severe COPD: a case control study of MMPs-1, 9 and 12 in a European population.

BACKGROUND: Genetic factors play a role in chronic obstructive pulmonary disease (COPD) but are poorly understood. A number of candidate genes have been proposed on the basis of the pathogenesis of COPD. These include the matrix metalloproteinase (MMP) genes which play a role in tissue remodelling and fit in with the protease--antiprotease imbalance theory for the cause of COPD. Previous genetic studies of MMPs in COPD have had inadequate coverage of the genes, and have reported conflicting associations of both single nucleotide polymorphisms (SNPs) and SNP haplotypes, plausibly due to under-powered studies. METHODS: To address these issues we genotyped 26 SNPs, providing comprehensive coverage of reported SNP variation, in MMPs- 1, 9 and 12 from 977 COPD patients and 876 non-diseased smokers of European descent and evaluated their association with disease singly and in haplotype combinations. We used logistic regression to adjust for age, gender, centre and smoking history. RESULTS: Haplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), showed an association with severe/very severe disease, corresponding to GOLD Stages III and IV. CONCLUSIONS: Those with the common A-A haplotype for these two SNPs were at greater risk of developing severe/very severe disease (p = 0.0039) while possession of the minor G variants at either SNP locus had a protective effect (adjusted odds ratio of 0.76; 95% CI 0.61 - 0.94). The A-A haplotype was also associated with significantly lower predicted FEV1 (42.62% versus 44.79%; p = 0.0129). This implicates haplotypes of MMP-12 as modifiers of disease severity.

Genetic predisposition to hypertension is associated with preeclampsia in European and Central Asian women.

Preeclampsia is a serious complication of pregnancy, affecting both maternal and fetal health. In genome-wide association meta-analysis of European and Central Asian mothers, we identify sequence variants that associate with preeclampsia in the maternal genome at ZNF831/20q13 and FTO/16q12. These are previously established variants for blood pressure (BP) and the FTO variant has also been associated with body mass index (BMI). Further analysis of BP variants establishes that variants at MECOM/3q26, FGF5/4q21 and SH2B3/12q24 also associate with preeclampsia through the maternal genome. We further show that a polygenic risk score for hypertension associates with preeclampsia. However, comparison with gestational hypertension indicates that additional factors modify the risk of preeclampsia.

A polymorphism of the alpha1-antitrypsin gene represents a risk factor for liver disease.

UNLABELLED: Alpha(1)-antitrypsin deficiency (AATD) due to homozygosity of the protease inhibitor (Pi) Z variant predisposes to childhood liver disease and pulmonary emphysema. About 10% of all neonates with AATD develop liver disease, and about 3% overall progress to severe disease. AATD is a principal genetic indication for liver transplantation in children. The liver pathology is associated with accumulation of abnormally folded protein in hepatocytes, the principal producers of circulating alpha(1)-antitrypsin (AAT). It is currently unknown why only a small proportion of Pi ZZ individuals progress to clinically significant cirrhosis. The AAT gene shows significant variation, and we hypothesized that cryptic genetic variants within the AAT gene may contribute to susceptibility to liver disease. In a case-control study consisting of 42 patients with established moderate-to-severe liver disease and 335 homozygous Pi ZZ patients who mostly presented with chronic obstructive pulmonary disease (n = 322: 242 index cases and 80 unaffected sibs) or were asymptomatic (n = 13) with no evidence of liver disease, we identified a single nucleotide polymorphism (SNP) that conferred a significant risk for liver disease (P = 0.007). The frequency of the SNP was no different in 242 Pi ZZ cases with chronic obstructive pulmonary disease compared with 80 nonindex cases. The SNP therefore appears to confer susceptibility to liver disease, although reporter gene assays failed to show any functional differences between alleles. CONCLUSION: This is the first description of a genetic modifier of liver disease in homozygous ZZ children and has potential implications for screening and possible therapies that are currently being developed.

The new genetics and chronic obstructive pulmonary disease.

Epidemiological and family studies provide evidence for genetic factors contributing to chronic obstructive airways disease (COPD) susceptibility. Studies to date have focused on candidate genes implicated in the pathogenesis of the disease. In general, many of these studies have been underpowered or have not been extensive enough in investigating the full extent of genetic variation in these genes. This has resulted in conflicting data with potential false positives or findings that have not been replicated. More recently, larger studies and extensive coverage of candidate genes have implicated genetic variants that may contribute to the disease. The use of unbiased genome-wide association studies offer the prospect of identifying new genes involved in COPD susceptibility and genetic modifiers of disease phenotypes. There is cause for optimism as a number of major complex diseases have been successfully tackled in this way. The review will highlight what has been achieved by genetic studies to date, some of the related problems and the future impact of high throughput technologies such as genome-wide association studies on our understanding of the genetic basis of COPD.

SERPINA3 (aka alpha-1-antichymotrypsin).

Recent advances in proteomic, transcriptomic and genomic technologies have revealed much about the ACT protein and gene. In this review, we summarize our current understanding of the structure and potential physiological roles of the ACT protein, catalogue the regulatory elements that have been implicated in expression of the ACT gene, describe its tissue-specific expression and list the single nucleotide polymorphisms (SNPs) within the gene that track ACT variability. The ACT gene has been implicated in a number of complex human disorders and its potential involvement as a risk factor for Alzheimer's disease has been the subject of intensive research. However, due to previous limitations in methodologies and inadequate sample numbers the data has been conflicting with many studies failing to be replicated. In this regard, we highlight some potential approaches, which may prove to be beneficial in future studies.

The hydroxy-methyl-glutaryl CoA reductase promoter polymorphism is associated with Alzheimer's risk and cognitive deterioration.

A link between cholesterol and Alzheimer's disease (AD) had been suggested. Hydroxy-methylglutaryl-coenzyme A reductase (HMGCR) is the rate limiting enzyme in the synthesis of cholesterol. A single nucleotide polymorphism (SNP) in the promoter of this gene, never described in Italian AD population, was investigated in case-control studies. Genotype distribution and allele frequency in two groups of AD patients and non demented controls were investigated. A cohort of AD patients were also followed up for 2 years, cognitive performances recorded and a possible influence of this SNP on the disease progression was tested. The CC genotype of the HMGCR gene was associated with a reduced risk of AD. Conversely the A allele of this polymorphism was over represented in AD patients. The presence of the A allele was also associated with an accelerated cognitive deterioration in AD patients followed up for 2 years. However, transfection experiments showed that this polymorphism did not directly influence functional activity in luciferase reporter gene assays. This polymorphism of the HMGCR gene appears to be linked to both AD risk and disease progression. Present findings reinforce the notion that abnormal regulation of cholesterol metabolism is a key factor in the pathogenesis of the disease.

Variants in the fetal genome near FLT1 are associated with risk of preeclampsia.

Preeclampsia, which affects approximately 5% of pregnancies, is a leading cause of maternal and perinatal death. The causes of preeclampsia remain unclear, but there is evidence for inherited susceptibility. Genome-wide association studies (GWAS) have not identified maternal sequence variants of genome-wide significance that replicate in independent data sets. We report the first GWAS of offspring from preeclamptic pregnancies and discovery of the first genome-wide significant susceptibility locus (rs4769613; P = 5.4 × 10-11) in 4,380 cases and 310,238 controls. This locus is near the FLT1 gene encoding Fms-like tyrosine kinase 1, providing biological support, as a placental isoform of this protein (sFlt-1) is implicated in the pathology of preeclampsia. The association was strongest in offspring from pregnancies in which preeclampsia developed during late gestation and offspring birth weights exceeded the tenth centile. An additional nearby variant, rs12050029, associated with preeclampsia independently of rs4769613. The newly discovered locus may enhance understanding of the pathophysiology of preeclampsia and its subtypes.

Altered transmission of maternal angiotensin II receptor haplotypes in fetal growth restriction.

Fetal growth restriction (FGR) predisposes to significant short- and long-term health problems. Epidemiological studies have suggested a role for inherited factors in its pathogenesis. The angiotensin II receptor genes, AGTR1 and AGTR2, are candidate genes because they mediate processes that are important for placentation. This study investigated AGTR1 and AGTR2 haplotypes and genotypes in FGR. A total of 107 families (father, mother, and baby) with FGR, and 101 families with normal pregnancies were genotyped at five sites in AGTR1 and six sites across AGTR2. All of the participants were white western Europeans. FGR was identified antenatally by ultrasound scans and confirmed postnatally by correcting the birth weight centile for gestation, infant sex, maternal height, weight, and parity. Fetal genes were investigated using transmission disequilibrium testing (TDT), and a case-control comparison of maternal haplotypes was conducted. FGR was associated with maternal (but not paternal) transmission of the AGTR1 haplotype (GenBank AF245699.1) g.4955T, g.5052T, g.5245C, g.5612A, and haplotype g.4955T, g.5052T, g.5245T, g.5612A. Haplotype g.4955A, g.5052G, g.5245T, g.5612G was undertransmitted (P = 0.002). TDT of the AGTR1 genotype showed undertransmission of maternal AGTR1 genotypes g.4955T>A (odds ratio (OR), 0.34 (95% confidence interval (CI), 0.14-0.86); P = 0.02), g.5052T>G (OR, 0.18 (0.06-0.48); P<0.001), and g.5612A>G (OR, 0.21 (0.08-0.55); P < 0.001) in FGR. There were no differences in maternal haplotype frequencies between normal pregnancy and FGR for AGTR1 or AGTR2 (P > 0.10). This is the first study to show distortion of transmission of maternal AGTR1 haplotypes in FGR, which suggests that this gene plays a role in FGR. In particular, maternal-fetal gene sharing may be an important factor.

Cryptic haplotypes of SERPINA1 confer susceptibility to chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a major cause of mortality and morbidity worldwide. While cigarette smoking is a major cause of COPD, only 15% of smokers develop the disease, indicating major genetic influences. The most widely recognized candidate gene in COPD is SERPINA1, although it has been suggested that SERPINA3 may also play a role. To detect cryptic genetic variants that might contribute to disease, we identified 15 SNP haplotype tags from high-density SNP maps of the two genes and evaluated these SNPs in the largest case-control genetic study of COPD conducted so far. For SERPINA1, six newly identified haplotypes with a common backbone of five SNPs were found to increase the risk of disease by six- to 50-fold, the highest risk of COPD reported to date. In contrast, no haplotype associations for SERPINA3 were identified.

Association of improved pulmonary phenotype in Irish cystic fibrosis patients with a 3' enhancer polymorphism in alpha-1-antitrypsin.

Modifier genes other than CFTR are thought to influence lung disease phenotype in cystic fibrosis (CF). In this study, we investigated the relationship between a polymorphism (1237 G --> A) in the 3' enhancer region of the alpha-1-antitrypsin (AAT) gene and pulmonary disease severity in 320 CF patients recruited from two independent adult referral centers in Ireland, and evaluated the in vivo effect of the polymorphism on AAT levels during acute infection. When corrected for confounding variables, the polymorphism was found to make a small but significant contribution to variance in percent predicted forced expired volume in 1 sec (FEV1) (1.1%, P = 0.05), with possession of the A allele being associated with better pulmonary function (AA/AG genotype: percent predicted FEV1, 70.8 +/- 3.9; GG genotype: percent predicted FEV1, 62.0 +/- 1.4). As would be expected of a modifier effect, the influence of the polymorphism was more marked in patient groups traditionally associated with more severe lung disease, contributing 3.2% (P = 0.033) to the variance in percent predicted FEV1 in patients homozygous for DF508, 3.3% (P = 0.007) to those infected with Pseudomonas aeruginosa, and 3% (P = 0.024) in female patients. In each instance, a positive association between possession of the A variant and higher percent predicted FEV1 was observed. We did not, however, find any evidence that possession of the A allele effected upregulation of AAT during acute infection in vivo. This lack of a demonstrable functional effect in vivo suggests that the polymorphism is a marker for a modifying effect on pulmonary phenotype in the Irish CF population by a mechanism that is yet to be explained.

A new promoter polymorphism in the alpha-1-antichymotrypsin gene is a disease modifier of Alzheimer's disease.

Increased levels of alpha-1-antichymotrypsin (ACT), a protease inhibitor and an acute phase protein, have been found in the brain and peripheral blood of patients with Alzheimer's disease (AD). Patients from northern Italy with a clinical diagnosis of probable AD, and patients with early onset AD (EOAD) from UK with AD neuropathological diagnosis were genotyped for a new polymorphism in the promoter region of the ACT gene which has been shown to affect ACT expression. A subset of patients with clinical AD from northern Italy was also followed up for 2 years and monitored for cognitive decline. The ACT TT promoter genotype was associated with an increased risk of EOAD independently from the presence of the apolipoprotein E (APOE) epsilon 4 allele. After manifestation of the disease the ACT TT genotype was also associated with faster cognitive decline in patients with the APOE allele epsilon 4. The ACT gene appears to influence the early clinical development of the disease, and the interaction of the ACT and APOE genes affects clinical progression of AD.