Sally Chappell

Searching for genetic clues to the causes of pre-eclampsia

Pre-eclampsia and its related syndromes are significant causes of maternal and fetal death, but much remains unclear about the underlying disease mechanisms. Epidemiological research has consistently demonstrated a familial predisposition to pre-eclampsia, which has encouraged genetic research in this area. The goal is the discovery of susceptibility genes which will inform understanding of the pathophysiology of pre-eclampsia, and may prove to be targets for therapeutic or preventative strategies. This review examines the application of molecular technologies to the search for genetic clues in pre-eclampsia and emphasizes the importance of integrative approaches. The results of recent genome-wide linkage studies have been particularly encouraging, identifying a number of loci which merit closer examination. Candidate gene studies have proved less fruitful, generating conflicting and inconclusive results. Possible explanations and remedies for this deficiency are discussed with a view to stimulating closer collaboration between researchers in this field.

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

BackgroundGenetic 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.MethodsTo 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.ResultsHaplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), showed an association with severe/very severe disease, corresponding to GOLD Stages III and IV.ConclusionsThose 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.

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

Alpha1‐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 alpha1‐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. (HEPATOLOGY 2007.)

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.

The SERPINE2 Gene and Chronic Obstructive Pulmonary Disease

To the Editor: In the February 2006 issue of the Journal, DeMeo et al.1 identified SERPINE2 as a positional candidate gene for susceptibility to chronic obstructive pulmonary disease (COPD [MIM 606963]) and reported on the association of polymorphic variants of this gene with early-onset disease in a family-based study and with severe disease in a case-control study. With early prior information provided by the authors, we have independently tested for an association of the SERPINE2 gene with COPD in the largest case-control study reported to date. Our study consists of 1,018 COPD cases and 911 controls prospectively recruited from six European centers. We have provided details about the patients elsewhere.2 The study population was screened for genotypes at the Medical Research Council (United Kingdom) Gene Services Unit for five SNPs (table 1) in the SERPINE2 gene. All the SNPs evaluated were reported in the study by DeMeo et al. as associated with disease, with three of the five associated with disease in both the family and case-control study cohorts they assessed. Table 1.  LD between SERPINE2 SNPs Expressed as r2[Note] We examined linkage disequilibrium (LD) between the SNPs (table 1) and evaluated SNP and haplotype associations as described elsewhere.2 DeMeo et al. did not report specific LD values between SNPs or noncontiguous SNPs contributing to haplotypes. SNPs and genotype frequencies in the study population are shown in table 2. We found no significant deviation from Hardy-Weinberg equilibrium in frequencies for any of the SNPs. Table 2.  SERPINE2 Genotype and Allele Frequencies in Controls and COPD Cases We found no association between any of the SERPINE2 SNPs and disease, in examining both the allelic and genotype distributions, although our study was well powered to detect associations of the magnitude observed by DeMeo et al., and we would have expected to see these frequency differences with the SNPs that we studied. We also failed to find a relationship between any haplotypes of these SNPs and disease (data not shown). It was of interest that the allele and genotype frequencies observed in our control and patient groups were virtually identical to those observed in control subjects by DeMeo et al., indicating a common distribution of SERPINE2 variants in the European and North American populations studied. Our previous study has also shown that there is no evidence of population stratification in our sample. Patients evaluated in both the family-based and case-control studies reported by DeMeo et al. represent a severe subset of the disease spectrum. To determine whether the association with SERPINE2 noted by DeMeo et al. was related to disease severity, we also analyzed SNP allele and genotype frequencies in the subgroup of our patients with forced expiratory volume at 1 s ⩽45% (n=388), a group that represents severe disease, but we failed to observe any association. Our inability to replicate the observations of DeMeo et al. in a more highly powered case-control study may be related to differences in the disease phenotype of the patients studied, because our patients included those with and without emphysema. The possibility, however, that the associations reported by DeMeo et al. represent false-positive results must also be considered. In this respect, it is of note that, in the study by DeMeo et al., different associations were reported for SNPs that are in linkage disequilibrium with one another. For example, rs3795879 and rs3795877 have an r2 value of 1 in HapMap, yet different associations with quantitative spirometric phenotypes were reported for the family study. Similarly, rs1438831 and rs920251 are in complete LD, with an r2 value of 1 in HapMap and 0.95 in our study; however, in DeMeo et al.’s case-control study, the allele and genotype frequencies of rs920251 were found to be significantly associated with disease (P values of 0.015 and 0.011, respectively), whereas no similar association was observed for rs1438831. In both instances, the almost complete linkage between these pairs of SNPs would be expected to result in similar associations. These results underline the importance of replication in other large independent studies before SERPINE2 can be unequivocally assigned as a candidate gene for COPD. It is becoming apparent that, to detect modest genetic effects for complex diseases, several independent studies may be required and the data may need to be subjected to meta-analysis. For example, this approach has been used to study Alzheimer disease (see Alzheimers Association Web site). Similar approaches need to be adopted for COPD. It would also be helpful to have similar criteria adapted for phenotypic selection and to plan prospective studies on this basis.

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

BackgroundGenetic 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.MethodsWe 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.ResultsOur 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.ConclusionsThese 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.

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.

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

A link between cholesterol and Alzheimers 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.

Copy Number Variation of the Beta-Defensin Genes in Europeans: No Supporting Evidence for Association with Lung Function, Chronic Obstructive Pulmonary Disease or Asthma

Lung function measures are heritable, predict mortality and are relevant in diagnosis of chronic obstructive pulmonary disease (COPD). COPD and asthma are diseases of the airways with major public health impacts and each have a heritable component. Genome-wide association studies of SNPs have revealed novel genetic associations with both diseases but only account for a small proportion of the heritability. Complex copy number variation may account for some of the missing heritability. A well-characterised genomic region of complex copy number variation contains beta-defensin genes (DEFB103, DEFB104 and DEFB4), which have a role in the innate immune response. Previous studies have implicated these and related genes as being associated with asthma or COPD. We hypothesised that copy number variation of these genes may play a role in lung function in the general population and in COPD and asthma risk. We undertook copy number typing of this locus in 1149 adult and 689 children using a paralogue ratio test and investigated association with COPD, asthma and lung function. Replication of findings was assessed in a larger independent sample of COPD cases and smoking controls. We found evidence for an association of beta-defensin copy number with COPD in the adult cohort (OR = 1.4, 95%CI:1.02–1.92, P = 0.039) but this finding, and findings from a previous study, were not replicated in a larger follow-up sample(OR = 0.89, 95%CI:0.72–1.07, P = 0.217). No robust evidence of association with asthma in children was observed. We found no evidence for association between beta-defensin copy number and lung function in the general populations. Our findings suggest that previous reports of association of beta-defensin copy number with COPD should be viewed with caution. Suboptimal measurement of copy number can lead to spurious associations. Further beta-defensin copy number measurement in larger sample sizes of COPD cases and children with asthma are needed.

Effects of Alzheimer's peptide and alpha 1-antichymotrypsin on astrocyte gene expression

We employed gene array technology to investigate the effects of alpha1-antichymotrypsin (ACT), soluble or fibrillar Alzheimers peptide (Abeta(1-42)) alone and the combination of ACT/Abeta(1-42) on human astrocytes. Using a 1.2-fold change as significance threshold, 398 astrocyte genes showed altered expression in response to these treatments compared to controls. Of the 276 genes affected by the ACT/soluble Abeta(1-42) combination, 195 (70.6%) were suppressed. The ACT/fibrillar Abeta(1-42) combination affected expression of 64 genes of which 58 (90.5%) were up-regulated. The most prominent gene expression changes in response to the ACT/soluble Abeta(1-42), were the down-regulation of at least 60 genes involved in transcription, signal transduction, apoptosis and neurogenesis. The ACT/fibril Abeta(1-42) increased the expression of genes involved in transcription regulation and signal transduction. Surprisingly, gene expression of astrocytes exposed to soluble or fibrillar Abeta(1-42) alone was largely unaffected. Thus, the molecular forms generated by the combination of ACT/Abeta(1-42) alter expression of astrocyte genes more profoundly in breadth and magnitude than soluble or fibrillar Abeta(1-42) alone, suggesting that pathogenic effects of Abeta(1-42) may occur as a consequence of its association with other proteins.