Noor Kalsheker

A Comprehensive Evaluation of Potential Lung Function Associated Genes in the SpiroMeta General Population Sample

Rationale Lung function measures are heritable traits that predict population morbidity and mortality and are essential for the diagnosis of chronic obstructive pulmonary disease (COPD). Variations in many genes have been reported to affect these traits, but attempts at replication have provided conflicting results. Recently, we undertook a meta-analysis of Genome Wide Association Study (GWAS) results for lung function measures in 20,288 individuals from the general population (the SpiroMeta consortium). Objectives To comprehensively analyse previously reported genetic associations with lung function measures, and to investigate whether single nucleotide polymorphisms (SNPs) in these genomic regions are associated with lung function in a large population sample. Methods We analysed association for SNPs tagging 130 genes and 48 intergenic regions (+/−10 kb), after conducting a systematic review of the literature in the PubMed database for genetic association studies reporting lung function associations. Results The analysis included 16,936 genotyped and imputed SNPs. No loci showed overall significant association for FEV1 or FEV1/FVC traits using a carefully defined significance threshold of 1.3×10−5. The most significant loci associated with FEV1 include SNPs tagging MACROD2 (P = 6.81×10−5), CNTN5 (P = 4.37×10−4), and TRPV4 (P = 1.58×10−3). Among ever-smokers, SERPINA1 showed the most significant association with FEV1 (P = 8.41×10−5), followed by PDE4D (P = 1.22×10−4). The strongest association with FEV1/FVC ratio was observed with ABCC1 (P = 4.38×10−4), and ESR1 (P = 5.42×10−4) among ever-smokers. Conclusions Polymorphisms spanning previously associated lung function genes did not show strong evidence for association with lung function measures in the SpiroMeta consortium population. Common SERPINA1 polymorphisms may affect FEV1 among smokers in the general population.

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimers disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case–control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer’s disease in seven independent case–control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer’s disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer’s disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer’s disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Amyloid precursor protein (APP) and the biology of proteolytic processing: relevance to Alzheimer's disease

The processing of amyloid precursor protein (APP) generates amyloid-beta (Abeta) peptides 1-40 and 1-42. The latter is neurotoxic and its accumulation results in amyloid fibril formation and the generation of senile plaques, the hallmark of Alzheimers disease (AD). Whilst there has been considerable progress made in understanding the generation of Abeta by alpha-, beta- and gamma-secretase activity on APP, recently enzymes involved in the degradation of Abeta have been identified including neprilysin and insulin-degrading enzyme (IDE). We review the pathways involved in proteolytic processing of APP and discuss the potential implications of aberrant proteolysis on neurodegeneration. It is conceivable that single nucleotide polymorphisms (SNPs) in the regulatory regions of genes in these proteolytic cascades, which alter their expression, could contribute to some of the age-related changes seen in AD.

Serpins: structure, function and molecular evolution

The superfamily of serine proteinase inhibitors (serpins) are involved in a number of fundamental biological processes such as blood coagulation, complement activation, fibrinolysis, angiogenesis, inflammation and tumor suppression and are expressed in a cell-specific manner. The average protein size of a serpin family member is 350-400 amino acids, but gene structure varies in terms of number and size of exons and introns. Previous studies of all known serpins identified 16 clades and 10 orphan sequences. Vertebrate serpins can be conveniently classified into six sub-groups. We provide additional data that updates the phylogenetic analysis in the context of structural and functional properties of the proteins. From these, we can conclude that the functional classification of serpins relies on their protein structure and not on sequence similarity.

A novel monoclonal antibody to characterize pathogenic polymers in liver disease associated with α1‐antitrypsin deficiency

Alpha1‐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 α1‐antitrypsin. This antibody was used to characterize the Z variant and a novel shutter domain mutant (His334Asp; α1‐antitrypsin Kings) identified in a 6‐week‐old boy who presented with prolonged jaundice. His334Asp α1‐antitrypsin rapidly forms polymers that accumulate within the endoplasmic reticulum and show delayed secretion when compared to the wild‐type M α1‐antitrypsin. The 2C1 antibody recognizes polymers formed by Z and His334Asp α1‐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 α1‐antitrypsin. Conclusion: Z and shutter domain mutants of α1‐antitrypsin form polymers with a shared epitope and so are likely to have a similar structure. HEPATOLOGY 2010

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.

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.

Pre‐eclampsia and the angiotensinogen gene

It is widely recognised that pre-eclampsia, one of the commonest and most serious complications of pregnancy, has an inherited component. Alterations in the reninangiotensin system in pregnancy suggest that genes encoding components of this system may be implicated. Recently, this suggestion has received support from genetic studies. In one study of affected siblings, there was distortion of allele sharing at a highly polymorphic dinucleotide repeat region close to the angiotensinogen gene (Arngrimsson et al. 1993). Another study demonstrated an association between an angiotensinogen gene variant and the disease (Ward et al. 1993). The second exon of the angiotensinogen gene, located on chromosome 1, encodes the signal peptide and the first 252 amino acids of the protein. Near the 3’ end of exon 2, a single base mutation (T-C) results in the substitution of threonine for methionine at amino acid 235. An association between the T235 variant and pre-eclampsia was described by Ward et al. (1993) in Caucasian women from Utah in the United States and in Japanese women. In these women possession of the T235 allele was also associated with higher plasma angiotensinogen concentrations. In our population, the frequency of the T235 allele among 137 healthy nonpregnant women (0.41) was found to correspond closely to that previously reported for a Caucasian population (Jeunemaitre et al. 1992). In this preliminary study we have now determined the frequency with which the T235 allele was found in 30 nulliparous pregnant Caucasian women in whom plasma angiotensinogen concentrations were also measured. Fifteen women were normotensive throughout pregnancy and the puerperium (median blood pressure 115/70 mmHg). Fifteen women developed pre-eclampsia according to strict criteria (median blood pressure 143/95 mmHg; minimum proteinuria 0.3 g/l). All women had been normotensive before

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.

Maternal and fetal angiotensinogen gene allele sharing in pre-eclampsia

Objective To compare the angiotensinogen genotypes in normotensive and pre‐eclamptic pregnancies in maternal and fetal samples.